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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Significant methodological deficiencies. Actual measurements of test concentrations were not performed. The test item is quite volatile, thus the exposure unclear.
Qualifier:
according to guideline
Guideline:
other: The procedures for static bioassay used were described in Methods of Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians, Environmental Protection Agency, Ecological Research Series EPA¬660/3-75-009, April 1975
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
Due to volatilization the actual exposure is unclear, as no analytical dose verification was performed.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
Dimethylformamide (DMF) (1.5 mL) was added to each sample to increase dispersion of the compound in the dilution water.
Appropriate weights of test compound were then added directly to the test chambers. All test concentrations were corrected for sample purity. The solvent control chamber received a 1.5-mL aliquot of DMF, which was equivalent to the highest amount used in any test solution.
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
TEST ORGANISM
- Common name: Bluegill sunfish
- Strain: ABC Lot #986
- Source: Osage Catfisheries in Osage Beach, MO, U.S.A.
- Length at study initiation (length definition, mean, range and SD): 27 (±1.9) mm
- Weight at study initiation (mean and range, SD): 0.50 (±0.11) g

ACCLIMATION
All test fish were held in culture tanks on a 16-hour daylight photoperiod and observed for at least fourteen days prior to testing. Fish culture techniques used were basically those described by Brauhn & Schoettger (1975). A daily record of fish observations was maintained. During acclimation, the fish received a standard commercial fish food supplemented with brine shrimp nauplii (Artemia sp.) daily until 48-96 hours prior to testing at which time feeding was discontinued.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
24, 48 and 96 h
Hardness:
40-45 mg/L as CaCO3
Test temperature:
22±1.0 °C
pH:
7.2 to 7.6 initially, at 48 and 96 hours pH was 6.8-7.3
Dissolved oxygen:
9.6 mg/L initially, at 48 and 96 hours, dissolved oxygen was 5.8-7.2 mg/L
Dissolved oxygen saturation at 22 °C is 8.8 mg/L
Nominal and measured concentrations:
Based on the results of preliminary testing, five concentrations of the test compound, ranging in a logarithmic series from 10 to 100 mg/L: 10, 18, 32, 56 and 100 mg/L were used. Also included were a dilution water control and a solvent control chamber.
Details on test conditions:
TEST SYSTEM
- Test vessel: Five-gallon glass vessels containing 15 liters of soft reconstituted water
- No. of organisms per vessel: 10
- Biomass loading rate: 0.33 g/L
The fish were added to the test chambers by random assignment within 30 minutes after addition of test material.


EFFECT PARAMETERS MEASURED:
All test organisms were observed once every 24 hours for mortality and abnormal (sub-lethal) effects. Any dead individuals were removed from the test chambers after each 24-hour observation.

A preliminary range-finding test of 72 hours duration was conducted to determine the concentration range for the definitive study. The preliminary test concentrations were 1, 10 and 100 mg/L.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Details on results:
The 24-, 48- and 96-hour LC50s were 75 mg/L for all three exposure periods. All results were based on the nominal concentrations of 10, 18, 32, 56 and 100 mg/L. The abnormal effects of mortality, surfacing, loss of equilibrium, dark discoloration, fish on the bottom of test chamber and/or quiescence were observed in all exposure periods at a test concentration of 18 mg/L and greater. The 96-hour no-observed effect concentration was estimated to be 10 mg/l, the lowest concentration tested. However, the actual test concentrations are questionable and the effects may be assigned to significantly lower exposure.
Reported statistics and error estimates:
Statistical analysis of the concentration versus effect data (generally mortality) was obtained by employing a computerized LC50 program developed by Stephan et al. The LC50 and its 95 percent confidence limits were calculated using the binomial, the moving average and the probit tests. However, if no mortality occurred or if a dose-response could not be demonstrated over a reasonable range (<37 to >63%), an LC50 and/or its 95 percent confidence limits could not be calculated. Three different methods of analyzing the data were used since no one method of analysis is appropriate for all possible sets of data that may be obtained. The method of calculation selected for presentation in this report was that which gave the narrowest confidence limits for the LC50, although all three models are valid.
Sublethal observations / clinical signs:

The test protocol noted that the concentration of volatile materials in the water should be confirmed by analytical techniques at 0 and 96 hours. However, there was no chemical analysis performed to verify or validate the test concentration. Owing to the demonstrated volatility of 1,2,3-trichloropropane, a 48-hour water evaporation study was performed to determine the actual concentrations after a 48-hour period. This analysis was performed in July 2001.

Validity criteria fulfilled:
yes
Remarks:
but not according to today's standards
Conclusions:
No valid endpoint can be derived from this study.
Executive summary:

The study was conducted in accordance with a recognized scientific method for determining acute toxicity to fish and in accordance with GLP regulations. There was no chemical analysis performed to verify or validate the test concentration. Owing to the demonstrated volatility of 1,2,3-trichloropropane, a 48-hour water evaporation study was performed to determine the degradation in test concentrations over a 48-hour period. These results verified that the test material is a volatile compound for which actual concentrations must be measured throughout the 96-hour study. The data do not provide sufficient information to support the conclusion regarding the 24-, 48- and 96-hour LC50s.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
May 1986
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with significant methodological deficiencies: Actual measurements of test concentrations were not performed. The test item is quite volatile, thus the exposure unclear.
Qualifier:
according to guideline
Guideline:
other: Methods of Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians, Environmental Protection Agency, Ecological Research Series EPA¬660/3-75-009, April 1975.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
Due to volatilization the actual exposure is unclear, as no analytical dose verification was performed.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
Dimethylformamide (DMF) (1.5 mL) was added to each sample to increase dispersion of the compound in the dilution water.
Appropriate weights of test compound were then added directly to the test chambers. All test concentrations were corrected for sample purity. The solvent control chamber received a 1.5-mL aliquot of DMF, which was equivalent to the highest amount used in any test solution.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: ABC Lot #1086
- Source: Spring Creek Trout Hatchery (ABC Lot #1086) in Lewistown, MT, U.S.A.
- Length at study initiation: mean standard length 38 (±3.6) mm
- Weight at study initiation: he rainbow trout had a mean weight of 0.76 (±0.23) g

ACCLIMATION
All test fish were held in culture tanks on a 16-hour daylight photoperiod and observed for at least fourteen days prior to testing. Fish culture techniques used were basically those described by Brauhn & Schoettger (1975). A daily record of fish observations was maintained. During acclimation, the fish received a standard commercial fish food supplemented with brine shrimp nauplii (Artemia sp.) daily until 48-96 hours prior to testing at which time feeding was discontinued.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
24, 48 and 96 hours
Hardness:
40-45 mg/L as CaCO3
Test temperature:
The test vessels were kept in a water bath at 12 °C (±1.0).
pH:
7.2 to 7.6 initially, at 48 and 96 hours pH was 7.0-7.2
Dissolved oxygen:
9.6 mg/L initially, at 48 and 96 hours, dissolved oxygen was 8-8.9 mg/L.
Dissolved oxygen saturation at 13 °C is 10.6 mg/L, and at 12 °C it is 10.8 mg/L.
Nominal and measured concentrations:
Based on the results of preliminary testing, five concentrations of the test compound, ranging in a logarithmic series from 10 to 100 mg/L: 10, 18, 32, 56 and 100 mg/L. Also included were a dilution water control and a solvent control chamber.
Details on test conditions:
TEST SYSTEM
- Test vessel Five-gallon glass vessels containing 15 liters of soft reconstituted water
- No. of organisms per vessel: 10
- Biomass loading rate: 0.51 g/L
The fish were added to the test chambers by random assignment within 30 minutes after addition of test material.

EFFECT PARAMETERS MEASURED:
All test organisms were observed once every 24 hours for mortality and abnormal (sub-lethal) effects. Any dead individuals were removed from the test chambers after each 24-hour observation.

A preliminary range-finding test of 72 hours duration was conducted to determine the concentration range for the definitive study. The preliminary test concentrations were 1, 10 and 100 mg/L.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
>= 56 - <= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Remarks on result:
other: 95% confidence limits
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
64 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
>= 32 - <= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Remarks on result:
other: 95% confidence limits
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
>= 32 - <= 56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Remarks on result:
other: 95% confidence limits
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks:
and abnormal effects
Remarks on result:
other: estimated
Details on results:
The 96-hour no-observed effect concentration was estimated to be <10 mg/L, the lowest concentration tested. The abnormal effects of mortality, surfacing, loss of equilibrium, dark discoloration, fish on the bottom of test chamber and/or quiescence were observed in all exposure periods and at concentrations of 18 mg/L or greater. At 10 mg/L, at 24 hours, only one fish displayed “surfacing.” All other observations were “normal” at 10 mg/L for 48-, 72- and 96-hour time periods. However, the actual test concentrations are questionable.
Reported statistics and error estimates:
Statistical analysis of the concentration versus effect data (generally mortality) was obtained by employing a computerized LC50 program. The LC50 and its 95 percent confidence limits were calculated using the binomial test. However, if no mortality occurred or if a dose-response could not be demonstrated over a reasonable range (<37 to >63%), an LC50 and/or its 95 percent confidence limits could not be calculated.
Sublethal observations / clinical signs:

The report noted that the 24-, 48- and 96-hour LC50s were 75, 64 and 42 mg/L, respectively. All values were based on the nominal concentrations of 10, 18, 32, 56 and 100 mg/L. The 96-hour no-observed effect concentration was estimated to be <10 mg/L, the lowest concentration tested. The abnormal effects of mortality, surfacing, loss of equilibrium, dark discoloration, fish on the bottom of test chamber and/or quiescence were observed in all exposure periods and at concentrations of 18 mg/L or greater. At 10 mg/L, at 24 hours, only one fish displayed “surfacing.” All other observations were “normal” at 10 mg/L for 48-, 72- and 96-hour time periods. However, the actual test concentrations are questionable.

Validity criteria fulfilled:
yes
Remarks:
but not according to today's standards
Conclusions:
No valid endpoint can be derived from this study.
Executive summary:

The study was conducted in accordance with a recognized scientific method for determining acute toxicity to fish and in accordance with GLP regulations. There was no chemical analysis performed to verify or validate the test concentration. Owing to the demonstrated volatility of 1,2,3-trichloropropane, a 48-hour water evaporation study was performed to determine the degradation in test concentrations over a 48-hour period. These results verified that the test material is a volatile compound for which actual concentrations must be measured throughout the 96-hour study. The data do not provide sufficient information to support the conclusion regarding the 24-, 48- and 96-hour LC50s.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
study completed in 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards and is described in sufficient detail.
Qualifier:
according to guideline
Guideline:
EPA OPP 72-1 (Fish Acute Toxicity Test)
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
The analyses included one spike and one duplicate sample for every 6 to 12 water samples.
Vehicle:
no
Details on test solutions:
A stock solution was prepared with a concentration of 533 mg 1,2,3-trichloropropane per liter (liq-liq equil). Hereafter five concentrations of the test compound, ranging in a logarithmic series from 19.1 to 107 mg/L, were prepared.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Source: cultured at the U.S. EPA Environmental Research Laboratory-Duluth, MN, U.S.A.
- Age at study initiation: 31 – 32 days
- Length at study initiation: 17.9 (± 1.294) mm
- Weight at study initiation: 0.077 (± 0.022) g
- Feeding: The fish were not fed 24 hr before and during the test.

ACCLIMATION
Adult fish were held at 25°C in flowing water with a controlled photoperiod of 16-hr light. They were fed frozen adult brine shrimp (Artemia sp.).
Only groups of fish having healthy appearance and no history of unusual thermal exposure or abnormally high mortality rate were used for toxicant exposure.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
44.1 ±0.48 mg/L
Test temperature:
24.6±0.12 °C
pH:
7.4 – 8.2
Dissolved oxygen:
7.9±0.2 mg/L
Nominal and measured concentrations:
nominal 19.1, 29.4, 45.2, 69.9, 107 mg/L
measured 16.4, 23.0, 33.9, 51.0, 86.7 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel fill volume: 2 L
- Renewal rate of test solution: 18 volume additions per day
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 1
- Biomass loading rate: 0.0428 g/L

EFFECT PARAMETERS MEASURED:
The number of dead fish was noted approximately every 24 hr after the beginning of the test, at which time they were also removed. Observations of fish behavior and toxic signs were made at 2-8, 24, 48, 72 and 96 hr, these observations were recorded on a checklist.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
66.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No 95% confidence limits could be calculated.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
65 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
behaviour
Remarks on result:
other: 95% confidence limits of 62.0 – 68.0 mg/L
Details on results:
At the highest test concentration all fish were dead after an exposure period of 24 hours. At the other test concentrations no mortality was observed. The 96-hour LC50 was 66.5 mg/L. No 95% confidence limits could be calculated. The 96-hour EC50 was 65.0 mg/L with 95% confidence limits of 62.0 – 68.0 mg/L. Affected fish lost schooling behavior, were hypoactive and underreactive to external stimuli, were darkly colored and lost equilibrium prior to death.
Reported statistics and error estimates:
The average weight of the individual fish was calculated by dividing the number of fish into the total sample weight.
The estimated LC50 and EC50 with corresponding 95% confidence intervals were calculated using the corrected average of the analyzed test chamber concentrations and the Trimmed Spearman-Karber Method (Hamilton et al 1977). The EC50s are based upon loss of equilibrium as manifested by the fish’s inability to maintain an upright position when swimming. The mean toxicant concentrations used in the calculations were corrected for analytical recoveries of spiked water samples. Confidence limits could not be calculated under the following conditions: lack of monotonically increasing deaths or effects with exposure concentrations, no complete (100%) effect concentration, no unaffected (0%) concentration and no concentration with a partial (>0 and <100%) effect.
Sublethal observations / clinical signs:

The results of the chemical analyses are given in the following table. All values were based on the corrected mean measured concentrations of 16.4, 23.0, 33.9, 51.0 and 86.7 mg/L.  

 

Nominal Concentration [mg/L]

Measured Concentration of TCP (mg/L)

Average concentration [mg/L]*

t = 0 hrs

t = 24 hrs

t = 48 hrs

t = 72 hrs

 

t = 96 hrs

0

< 0.5

< 0.5

< 0.5

< 0.5

< 0.5

< 0.5 (< 0.53)

19.1

12.6

16.7

16.5

16.7

15.2

15.5 (16.4)

29.4

17.8

23.6

22.7

23.6

21.3

21.8 (23.0)

45.2

27.3

34.4

34.0

34.4

30.9

32.2 (33.9)

69.6

41.9

50.4

49.6

52.7

47.4

48.4 (51.0)

107

67.4

85.8

84.0

91.4

82.8

82.3 (86.7)

* Between brackets, the corrected average concentrations are given. The mean toxicant concentrations used in the calculations were corrected for analytical recoveries of spiked water samples. The recovery was 94.9 %

Validity criteria fulfilled:
yes
Conclusions:
The 96-h LC50 of TCP to Fathead minnows is 66.5 mg/L
Executive summary:

The study was performed under non-GLP conditions in accordance with a recognized scientific method for determining acute toxicity to fish (EPA OPP 72-1). Chemical analysis of the test item, TCP, was performed to verify the test concentration. In the publication acute toxicity tests with fathead minnows for several organic compounds are described but detailed information about individual tests (e.g. dilution water, measurements of test conditions) is not given. Nonetheless the documentation is sufficient for assessment and the results are considered valid and conclusive.

The test animals used were (Pimephales promelas) were during 96 h exposed to corrected mean measured concentrations of 16.4, 23.0, 33.9, 51.0 and 86.7 mg/L.  Behaviour and mortality were recorded.

Based on mortality an LD50 of 66.5 mg/L was derived, while behavioural effects were observed at somewhat lover concentrations.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Poor documentation. Actual measurements of test concentrations were probably not performed. The test item is quite volatile, thus the exposure unclear.
Principles of method if other than guideline:
Fish were incubated in water with dissolved test item and the mortality was counted after 7 days. Poor documentation. Actual measurements of test concentrations were probably not performed. The test item is quite volatile, thus the exposure unclear.
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Poecilia reticulata
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Duration:
7 d
Dose descriptor:
LC50
Effect conc.:
41.6 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Validity criteria fulfilled:
not applicable
Conclusions:
The (sub)acute LC50 is assigned to 41.6 mg/L however the exposure is unclear.
Executive summary:

The derived endpoint is not a standard one being neither short, nor long-term observation related. Because of the lacking analytical dose control the exposure is uncertain. It may be the case that due to the test item volatilization only acute effects have been produced. The derived effect concentration is in the range of other short term observations, but constitutes the lowest value. Accordingly the reported LC50 of 41.6 mg/L is used for CSA. Further evaluation is considered not insightful because the fish are not the most susceptible aquatic organism group.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conducted according to Japanese National Toxicology program standards. Only 48 h exposure.
Qualifier:
according to guideline
Guideline:
other: CSCL Japan standards
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Oryzias latipes
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
109 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Conclusions:
The study is not relevant for CSA.
Executive summary:

Due to the restriction of the exposure time to 48 hours the study does not provide additional evidence for the endpoint derivation. A valid and reliable study for 96 hour exposition exists and the LC50 concentration is lower.

Description of key information

TCP exhibits acute toxicity to fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
41.6 mg/L

Additional information

The available studies on acute toxicity to fish indicate significant species to species variability. Japanese medaka was fewest susceptible as evidenced by a 48-h LC50of 109 mg/L (MITI 1992). The study is valid and conclusive but the exposure time shorter than the standard. Geiger et al (1990) found a 96-h LC50 of 66.5 mg/L to fathead minnow (Pimephales promelas). The study is considered valid and conclusive and however it was not performed under GLP.

In further toxicity studies conducted with several fish species, the lowest LC50 value (7-day incubation, which does not mean exposure, see below) was established for guppies (Poecilia reticulata) to be 41.6 mg/L (Könemann 1981). For the rainbow trout (Oncorhynchus mykiss) and bluegill sunfish (Lepomis macrochirus) the 96-h LC50s were 42 and 75 mg/L respectively while the estimated NOEC value was <10 mg/L (Bowman 1986).

However these studies have methodological deficiencies with regard to the analytical dose verification they trigger the hazard assessment because the found the lowest effect levels. Loss due to evaporation of TCP from the aqueous phase was demonstrated in a study simulating a 48-h daphnid open vessel test (Solvay 2001, Internal Memorandum). Due to the non-verification of the actual exposure level only an underestimation the toxic effects is possible. In conclusion the LC50 of TCP is assessed to be 41.6 mg/L as the actual exposure to the evaporating test item may be not more than 96 h. This is considered an acceptable approximation of the short term toxicity to fish for assessment since fish are obviously not the most sensitive taxonomic group.

Because good long term observations form fish are available further testing on fish acute effects is beside the point.