New Jersey Administrative Code
Title 7 - ENVIRONMENTAL PROTECTION
Chapter 18 - REGULATIONS GOVERNING THE CERTIFICATION OF LABORATORIES AND ENVIRONMENTAL MEASUREMENTS
Subchapter 7 - TOXICITY TESTING
Section 7:18-7.5 - Acute toxicity testing methodology

Universal Citation: NJ Admin Code 7:18-7.5

Current through Register Vol. 56, No. 18, September 16, 2024

(a) A laboratory shall not use an acute toxicity test experimental design unless it satisfies all applicable requirements of this section.

(b) When the purpose of a definitive acute toxicity test is to determine compliance with an LC[50] or EC[50] permit limitation, the test shall satisfy all of the following requirements:

1. The test shall include at least one control treatment, and a series of at least five effluent concentrations;

2. The laboratory shall perform each control treatment and each effluent concentration at least in duplicate, and shall conduct additional replicate series when necessary to achieve required test precision. The laboratory shall use only true replicates, with no water connections between test chambers;

3. If the toxicity of the effluent to the test organism is not known, the laboratory shall select concentrations that are evenly spaced on either a logarithmic scale or a geometric scale. The concentration of effluent in each treatment (except for the highest concentration and each control) shall be at least 50 percent of the next highest one;

4. If the toxicity of the effluent to the test organism is known approximately, the laboratory shall select concentrations of effluent that are evenly spaced (on either a logarithmic scale or geometric scale) around the expected LC[50] or EC[50]. Except for the highest concentration and each control(s), the test concentration shall be at least 60 percent of the next higher one. The use of a 100 percent effluent concentration is not required where the inclusion of such concentration is not within the expected range of the LC[50];

5. Every toxicity test shall include a dilution water control treatment consisting of the same dilution water, conditions, procedures, type and number of organisms as used in the effluent treatments, except that the laboratory shall add none of the effluent being tested to the dilution water. Whenever the laboratory uses artificial sea salts to adjust the salinity of either the dilution water sample or effluent sample, an additional control treatment shall be included. This additional control treatment shall consist of replicate chambers containing only artificial saltwater, made with the same artificial sea salts used to adjust the samples. The artificial saltwater shall be made to the same standardized salinity and pH as the other test treatments; and

6. The laboratory shall expose at least 20 test organisms to each effluent concentration and each control treatment. For example, when the laboratory is conducting the test in duplicate, it shall expose at least 10 organisms per test chamber. The number of organisms used in each effluent concentration shall be equal to the number used in other effluent concentrations and to the number used in the control.

(c) When the effluent is known to generally have an LC[50] of greater than or equal to 100 percent and the laboratory is conducting an N.M.A.T. definitive acute toxicity test for determining compliance with a "no measurable acute toxicity" permit limitation, the toxicity test design shall meet the following requirements:

1. The test series shall consist of at least one control treatment, and a series of at least five effluent concentrations;

2. The laboratory shall perform each control treatment and each effluent concentration at least in duplicate, and shall conduct additional replicate series when necessary to achieve required test precision. The laboratory shall use only true replicates, with no water connections between test chambers;

3. The laboratory shall expose at least 20 test organisms to each effluent concentration and each control treatment. For example, when the laboratory is conducting the test in duplicate, it shall expose at least ten organisms per test chamber. The number of organisms used in each effluent concentration shall be equal to the number used in other effluent concentrations and to the number used in the control; and

4. Every toxicity test shall include a dilution water control treatment consisting of the same dilution water, conditions, procedures, type and number of organisms as used in the effluent treatments, except that the laboratory shall add none of the effluent being tested to the control treatment. Whenever the laboratory uses artificial sea salts to adjust the salinity of either the dilution water sample or the effluent sample, an additional control treatment shall be included. This additional control treatment shall consist of replicate chambers containing only artificial saltwater, made with the same artificial sea salts used to adjust the samples. The artificial saltwater shall be made to the same standardized salinity as the other test treatments.

(d) When there is no historical aquatic toxicological data available on an effluent, the laboratory shall conduct a range-finding toxicity test to ascertain the range of effluent concentrations for subsequent definitive tests. The range-finding toxicity test shall satisfy the following requirements:

1. The range-finding toxicity test shall consist of one or more control treatments, and at least four treatments which shall include a 100 percent effluent-by-volume, 50 percent effluent-by-volume, and 12.5 percent effluent-by-volume. The laboratory shall use either a single test series or replicates;

2. Every range-finding test shall include a dilution water control treatment. This treatment shall consist of the same dilution water, conditions, procedures, type and number of organisms as used in the effluent treatment, except that none of the effluent being tested shall be added to the dilution water; and

3. Five or more test organisms shall be exposed to each control treatment and each effluent treatment.

(e) The laboratory shall conduct tests as either static, renewal, or flow-through tests in accordance with the following:

1. The laboratory shall conduct the following as either a renewal test or a flow-through test:
i. Any definitive toxicity test with cold-water fishes, warm-water fishes, saltwater fishes or saltwater macrocrustaceans; and

ii. Any N.M.A.T. or N.O.A.E.C. definitive toxicity test with cold-water fishes, warm-water fishes, saltwater fishes or saltwater macrocrustaceans;

2. The laboratory shall conduct as either a static test or a flow-through test any range-finding toxicity test with coldwater fishes, warmwater fishes, saltwater fishes or saltwater macrocrustaceans; and

3. The laboratory shall conduct the following as a static test:
i. Any definitive toxicity test with freshwater zooplankton;

ii. Any N.M.A.T. or N.O.A.E.C. definitive toxicity test with freshwater zooplankton; and

iii. Any range-finding toxicity test with freshwater zooplankton.

(f) The laboratory shall conduct toxicity tests for the durations described below:

1. Cladoceran range-finding toxicity test duration shall be at least 24 hours;

2. Cladoceran definitive toxicity test, N.O.A.E.C. and N.M.A.T. definitive toxicity test durations shall be 48 hours;

3. Mysid range-finding toxicity test duration shall be at least 24 hours;

4. Mysid definite toxicity test, N.O.A.E.C. and N.M.A.T. definitive toxicity test durations shall be at least 96 hours;

5. Grass shrimp range-finding toxicity test duration shall be at least 24 hours;

6. Grass shrimp definitive, N.O.A.E.C. and N.M.A.T. definitive toxicity test durations shall be at least 96 hours;

7. The duration of any range-finding toxicity test done with fishes shall be at least 24 hours; and

8. The duration of any definitive toxicity test with fishes or any N.M.A.T. or N.O.A.E.C. definitive toxicity test with fishes shall be at least 96 hours.

(g) Laboratories shall conduct toxicity tests with the test organisms randomly distributed to the test chambers by either of the two following methods:

1. Adding to each chamber no more than 20 percent of the total number to be assigned to each chamber, then repeating the process until each test chamber contains the total number of test organisms desired; or

2. Randomly assigning one test organism to each test chamber, then randomly assigning a second test organism to each test chamber, etc., continuing the random assignments until the total number of test organisms desired has been distributed to each test chamber.

(h) The laboratory shall maintain dissolved oxygen in the test chambers in accordance with the following requirements:

1. At all times during testing with cold-water fish species, the laboratory shall maintain dissolved oxygen at greater than 60 percent of saturation;

2. At all times during testing with other species, the laboratory shall maintain dissolved oxygen at greater than 40 percent of saturation;

3. In static and renewal acute toxicity tests, the laboratory shall gently aerate all test chambers if dissolved oxygen falls below 60 percent of saturation for the freshwater coldwater fishes or below 40 percent of saturation for the freshwater warmwater fishes and all saltwater species. If aeration is not going to be continuous, the laboratory shall stop aeration when dissolved oxygen reaches 100 percent of saturation for the freshwater coldwater fishes, or 60 percent of saturation for the freshwater warmwater fishes and all saltwater species;

4. In flow-through toxicity tests, the laboratory shall gently aerate all test chambers while maintaining the turnover rate if the dissolved oxygen falls below 70 percent of saturation for freshwater coldwater fishes, or below 50 percent of saturation for the freshwater warmwater fishes and all saltwater species. If aeration is not going to be continuous, the laboratory shall stop aeration when dissolved oxygen reaches 100 percent of saturation for the freshwater coldwater fishes or 60 percent of saturation for the freshwater warmwater fishes and for the saltwater species; and

5. In static testing with Cladoceran, the laboratory shall measure dissolved oxygen either on the test solutions that are used in the acute toxicity test, or on a duplicate series of test solutions not containing test organisms. The laboratory shall perform zero hour of exposure measurements either upon an aliquot of the solutions being dispensed to the test chambers, or upon a duplicate series of test solutions set up as another replicate without test organisms. The laboratory shall measure dissolved oxygen for all test concentrations and the control at zero (0) and 48 hours. The laboratory shall measure dissolved oxygen at 24 hours for those test concentrations where there is 100 percent mortality or immobilization. The laboratory shall not aerate Cladoceran test chambers under any circumstances, even if dissolved oxygen levels fall below 40 percent of saturation.

(i) The laboratory shall maintain test temperatures in accordance with the following requirements:

1. Freshwater organisms shall be tested at the following temperatures:
i. Cold-water fishes shall be tested at 12 degrees Celsius; and

ii. Warm-water fishes and freshwater zooplankton shall be tested at 20 degrees Celsius;

2. Saltwater organisms shall all be tested at 20 degrees Celsius.

3. The laboratory shall maintain the temperature of test solutions within +/- 2.0 degrees Celsius of the required test temperature.

(j) The laboratory shall test sheepshead minnow, inland silverside, grass shrimp, and Mysid at a salinity of five ppt to 32 ppt, +/-10 percent. The laboratory shall test the tidewater silverside and Atlantic silverside at a salinity of 15 ppt to 32 ppt, +/-10 percent. A standardized salinity should be 25 ppt, +/-one ppt for all saltwater organisms.

(k) The laboratory shall provide test organisms with light during testing in accordance with the following requirements:

1. The laboratory shall provide Cladoceran with wide spectrum light at an intensity of 50 to 100 foot candles, measured at the surface of the test chamber solutions. The photoperiod shall be a steady 16 hours light and 8 hours dark;

2. The laboratory shall provide Mysidopsis sp. and Palaemonetes pugio with wide spectrum light at an intensity of 50 to 100 foot candles measured at the surface of the test chamber solutions. The photoperiod shall be a steady 14 to 16 hours light and eight to 10 hours dark; and

3. The laboratory shall provide freshwater and saltwater fishes with wide spectrum light at an intensity of 50 to 100 foot candles, measured at the surface of the test chamber solutions. The photoperiod shall be a steady 14 to 16 hours light and eight to 10 hours dark.

(l) For static or renewal toxicity tests the test organism loading shall not exceed the following:

1. Loading of grass shrimp and coldwater, warmwater, and saltwater fishes shall not exceed 0.65 g/L of test solution;

2. Loading of Cladoceran shall not exceed one daphnid per 20 mL of test solution.

3. Loading of Mysids shall not exceed one mysid per 40 mL of test solution.

(m) For flow-through toxicity tests the test organism loading shall not exceed the following:

1. Loading of grass shrimp and coldwater, warmwater, and saltwater fishes shall not exceed five g/L of test solution; and

2. Loading of Mysids shall not exceed one mysid per 20 mL of test solution.

(n) The laboratory shall take organisms for use in testing only from groups that meet the requirements in 7:18-7.4(e)3 vi concerning mortality during acclimation and holding.

(o) When an effluent discharged to estuarine or marine waters consists of adulterated freshwater, the laboratory shall adjust the salinity of the effluent only in accordance with the following:

1. When using effluent concentrations greater than 75 percent effluent-by-volume, the laboratory shall adjust the salinity of the effluent test concentrations by using artificial sea salts. In the case of effluent/dilution water mixtures, the laboratory shall add the salts to the effluent either before or after the effluent and dilution water sample aliquots are mixed;

2. When using effluent concentrations less than or equal to 75 percent effluent-by-volume, the laboratory shall adjust the salinity of the effluent test concentrations by using either a hypersaline brine that is prepared in compliance with 7:18-7.4(b)8, or dry artificial sea salts. The laboratory shall make the adjustments either before or after the effluent and dilution water samples are mixed; and

3. When a laboratory is using artificial sea salts to adjust the freshwater effluent salinity, and the pH of the test concentration to which the artificial sea salts were added drifts more than 0.5 pH units from the initial pH, the laboratory shall also adjust the pH of the test concentration to within 0.5 pH units of the pH of the original test concentration by using NaOH or HCl. The laboratory shall document and report adjustments and treatments of the effluent along with the test results. The laboratory shall include in the documentation the name and amount of reagent used to adjust the pH, and the pH before and after pH adjustment.

(p) To initiate a static or renewal test, the laboratory shall place the test organisms in the test chambers within 30 minutes after the effluent is added to the dilution water.

(q) To initiate a flow-through test, the laboratory shall place the test organisms in test chambers after the dilutor system has been calibrated, with the dilution water and effluent, and the test solutions have been flowing through the test chambers for a period of 24 hours at a rate which ensures five 90 percent replacements of water volume in each test chamber. During this period, the laboratory shall make all necessary adjustment to flow rate, temperature, and aerations.

(r) The laboratory shall feed test organisms during toxicity testing in accordance with 7:18-7.4(e)3 vii(15) and the following requirements:

1. The laboratory shall feed Cladoceran and freshwater coldwater fish up to but not during acute toxicity testing;

2. The laboratory shall feed Mysids and grass shrimp up to and during acute toxicity testing. During testing, the laboratory shall feed the Mysids and grass shrimp at a rate of 0.1 mL concentrated hatched Artemia per Mysid and grass shrimp twice daily, which is approximately 100 brine shrimp nauplii/Mysid/day; and

3. The laboratory shall feed warmwater freshwater and saltwater fish up to the beginning of the acute toxicity test. During testing, the laboratory shall feed the fish at a rate of 0.2 mL concentrated hatched Artemia two hours prior to the 48 hour test solution renewal.

(s) The laboratory shall collect biological data and make biological observations in accordance with the following requirements:

1. To determine the effluent's EC[50] in acute toxicity tests with Cladoceran, the laboratory shall observe and record the organisms' immobilization, defined as the inability to move the appendages when gently prodded;

2. To determine the effluent's EC[50] in acute toxicity tests with all fishes, mysids and grass shrimp, the laboratory shall measure death, defined as no movement of any kind, especially the absence of respiratory movements, and no reaction to gentle prodding;

3. The laboratory shall count the number of dead or affected organisms in each test chamber at each 24 hour exposure interval throughout the test and, to intercept potential problems, these observation should occur at least twice daily;

4. The laboratory shall remove dead organisms from test chambers at least each time dead or affected organisms are counted;

5. The laboratory shall observe the test organisms' appearance and behavior at least daily, and record the observations on the acute toxicity test bench sheets using the applicable terms or codes in Table 7.5(s).
TermExplanationCode
NormalUnaffected1
InactiveAbnormally low activity, motionless or2
nearly so, weak and enfeebled
IrritatedHyperactivity, muscle spasms, erratic3
swimming
SurfacingRising and remaining unusually long at4
the surface
Abnormal Body OrientationInverted or turned approximately 905
[degrees]; laterally from normal body
position
Abnormal Skin ColorLight discolored, dark discolor or6
varidiscolored (mottled)
Abnormal Skin ConditionMucus shedding or coagulations,7
hemorrhaging from gills, eyes or anal
opening
Abnormal RespirationRapid, slow, gulping or periodic flexure8
of the operculum of fish as to reverse
water flow (coughing)

6. The laboratory shall not stress live organisms when determining whether test organisms are dead, immobilized, or otherwise affected, and when removing dead organisms. Any movement of test chambers and any prodding shall be done very gently; and

7. The laboratory shall determine the weights and lengths of test fish and grass shrimp by weighing, measuring and discarding at least a 10 percent sample of the batch of organisms to be used in the test, or by weighing and measuring the surviving control organisms after the test. For test fish, the laboratory shall determine the total length; for grass shrimp, the laboratory shall measure from rostrum to telson.

(t) The laboratory shall collect and analyze chemical and physical data in accordance with the following requirements:

1. The laboratory shall analyze all chemical and physical parameters (excluding salinity) under this subchapter in accordance with the requirements set forth in 40 C.F.R. 136 and 7:18-5.3;

2. The laboratory shall compute salinity based on chlorinity, electrical conductivity, or refractive index;

3. If an effluent has exhibited, or is known to exhibit, a high dissolved oxygen demand, the laboratory shall monitor dissolved oxygen at the following frequency except as provided in (t)5 below:
i. For the first four hours of testing, once every two hours in all of the control and effluent test chambers; and

ii. After the first four hours of testing, at least once daily in each test chamber in which there are living test organisms;

4. If the effluent has not exhibited, nor is known to exhibit, a high dissolved oxygen demand, the laboratory shall monitor dissolved oxygen at least once every 24 hours in all of the control and effluent test chambers, except as provided in (t)5 below;

5. When testing with Cladoceran, the laboratory shall monitor dissolved oxygen in accordance with 7:18-7.5(h)5 above;

6. When a laboratory conducts a toxicity test in either a constant temperature area or water bath:
i. The laboratory shall measure and record the temperature of the area or bath when the test is initiated, at least once every six hours during the test, and upon termination of the test;

ii. When testing with Cladoceran, the laboratory shall measure and record the temperature in a blank test chamber (a chamber without test organisms), at least daily;

iii. When testing with fish, grass shrimp or Mysids, the laboratory shall measure and record the temperature in each test chamber, including the control exposure;

7. When a toxicity test is not conducted in a constant temperature area or water bath, the laboratory shall measure and record the temperature in at least one control test chamber and one effluent concentration test chamber at least hourly throughout the test. The laboratory shall measure and record the other test chamber temperatures at least once daily throughout the test;

8. A laboratory performing a static acute toxicity test shall collect the following chemical and physical data, in addition to the data described in (t)1 through 7 above:
i. In toxicity tests using freshwater dilution water, just before initiating the test the laboratory shall measure and record harness, pH, total residual chlorine (when detected initially in either the dilution water or effluent sample), and specific conductance. If the laboratory is performing definitive testing with Cladoceran, it shall also measure and record the above information upon the termination of the test. For all tests, the laboratory shall make these measurements on aliquots of the same test solutions used to set up the test initially; and

ii. In toxicity tests using saltwater dilution water, just before initiating the test the laboratory shall measure and record pH, salinity, and total residual chlorine (when detected initially in the effluent sample). For all tests, the laboratory shall make these measurements on aliquots of the same test solutions used to set up the test initially;

9. A laboratory performing a flow-through or renewal toxicity test shall collect the following chemical and physical data, in addition to the data described in (t)1 through 7 above:
i. In toxicity tests using a freshwater dilution water, the laboratory shall measure and record just before test initiation pH, hardness, total alkalinity, total residual chlorine (when detected initially in either the dilution water or effluent sample), and specific conductance. In addition, the laboratory shall measure and record the pH, and specific conductance once every 24 hours during the test, and upon termination of the test in at least one set of replicate chambers that includes the control and all effluent concentrations. Also once every 24 hours during the test, the laboratory shall measure and record the hardness, total alkalinity, and total residual chlorine (when detected initially in either the dilution water or effluent sample) once every 24 hours during the test, and upon termination of the test in at least one set of replicate chambers that includes the control and the highest effluent concentration. The laboratory shall take all the measurements either upon aliquots of the same test solutions or upon aliquots taken from the test chambers.

ii. In toxicity tests using saltwater dilution water, the laboratory shall measure and record just before test initiation pH, total alkalinity, total residual chlorine (when detected initially in either the dilution water or effluent sample), and salinity. In addition, the laboratory shall measure and record the pH and salinity once every 24 hours during the test, and upon termination of the test in at least one set of replicate chambers that includes the control and all effluent concentrations. Also once every 24 hours during the test, the laboratory shall measure and record the total alkalinity and total residual chlorine (when detected initially in either the dilution water or effluent sample) once every 24 hours during the test, and upon termination of the test in at least one set of replicate chambers that includes the control and the highest effluent concentration. The laboratory shall take all the measurements either upon aliquots of the same test solutions or upon aliquots taken from the test chambers.

(u) A laboratory conducting an N.O.A.E.C. definitive acute toxicity test for determining compliance with a "no observed adverse effect concentration" permit limitation shall design the toxicity test in accordance with the following requirements:

1. The laboratory shall include in the test at least one control treatment, and a series of at least five effluent concentrations;

2. The laboratory shall conduct the test with two replicates. The laboratory shall use only true replicates, with no water connections between test chambers;

3. The laboratory shall expose 10 test organisms per chamber to each effluent concentration;

4. The laboratory shall include in every toxicity test a dilution water control treatment consisting of the same dilution water, conditions, procedures, type and number of organisms as used in the effluent treatments, except that the laboratory shall add none of the effluent being tested to the control treatment. Whenever the laboratory uses artificial sea salts to adjust the salinity of either the dilution water sample or the effluent sample, an additional control treatment shall be included. This additional control treatment shall consist of replicate chambers containing only artificial saltwater, made with the same artificial sea salts used to adjust the samples. The artificial saltwater shall be made to the same standardized salinity as the other test treatments; and

5. The laboratory shall expose at least 20 test organisms to each control treatment and each effluent treatment.

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