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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:
key study
Study period:
28 March to 01 April 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
Commission Directive 92/69/EEC
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Verification of test concentrations took place at 0, 24 and 96 hours.
Vehicle:
no
Details on test solutions:
TEST WATER
- Laboratory tap water dechlorinated by addition of sodium thiosulphate.
- Typical water quality characteristics are shown in Appendix 3 (attached).
- Total hardness approximately 100 mg/L as CaCO3
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST SPECIES
- Source: Parkwood Trout Farm, Wigmore, Kent, UK
- Stock held since 04 January 1994 was acclimatised to test conditions between 21 March and 28 March 1994
- Stock fish were maintained in a glass fibre tank with a "single pass" water renewal system
- Lighting was controlled to give a cycle of 16 hours light and 8 hours darkness
- Temperature 14 °C
- Dissolved oxygen: ≥ 9.7 mg/L
- Feeding: Commercial trout pellets daily (discontinued 24 h prior to test)
- Mortalities: < 1 % in the 7 days prior to the start of the test
- Medication: None
- Mean standard length: 4.4 cm (SD = 0.4 cm)
- Mean weight: 1.28 g (SD = 0.32 g)




Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
Total hardness approximately 100 mg/L as CaCO3
Test temperature:
14 °C
pH:
7.4-7.5 (see Appendix 1, attached)
Dissolved oxygen:
9.9-10.2 mg/L (see Appendix 1, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
RANGE-FINDING STUDY
- Nominal test item concentrations of 1.0, 10 and 100 mg/L

DEFINITIVE TEST
- Nominal test item concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L
Details on test conditions:
METHOD OF PREPARATION
- Test material (2.00 g) was dispersed in water with the aid of ultrasonic disruption and the volume was adjusted to 1 L to give a 2.00 g/L stock solution.
- Aliquots of stock solution (10, 18, 32, 56 and 100 mL) were each separately dispersed in dechlorinated tap water and the volume was adjusted to 20 L to give the test series.

STABILITY OF TEST CONCENTRATIONS
- Ensured by daily renewal of the test media and verified by chemical analysis of the control and all surviving test groups at 0, 24 and 96 h (see Appendix 2, attached).
- Data relating to the identity, purity and stability of the test material were the responsibility of the sponsor.

RANGE FINDING STUDY
- Concentrations for the definitive test were assigned following a preliminary range-finding study in which 3 fish were exposed to three concentrations of test solution.

EXPOSURE CONDITIONS FOR DEFINITIVE TEST
- Test vessels: Glass aquaria holding 20 L of test media.
- Experimental design: Five test concentrations plus one control; ten animals per test concentration.
- Method of initiation: Fish were placed at random in prepared test media.
- Loading: 0.64 g bw/L.
- Photoperiod: 16 h light and 8 h dark.
- Aeration: Via narrow bore glass tubes.
- Medium renewal: Daily.
- Environmental measurements: pH, dissolved oxygen concentration and temperature were recorded daily throughout the study.
- Criteria of death: Absence of (i) respiratory movement and (ii) response to physical stimulation.

Duration:
3 h
Dose descriptor:
LC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
6 h
Dose descriptor:
LC50
Effect conc.:
7.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits
Remarks:
5.6-10 mg/L
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits
Remarks:
1.8-3.2 mg/L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits
Remarks:
1.8-3.2 mg/L
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits
Remarks:
1.8-2.5 mg/L
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits
Remarks:
1.4-2.0 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
RANGE FINDING STUDY
- No mortalities were observed at a test solution concentration of 1.0 mg/L
- Mortalities were observed at test solution concentrations of 10 and 100 mg/L and these findings were used to indicate the choice of concentration for the definitive test.

VERIFICATION OF TEST CONCENTRATIONS
- Chemical analysis showed the measured test concentrations to be near nominal and it was considered justifiable to calculate LC50 values in terms of nominal test concentrations only.

DEFINITIVE TEST
- Cumulative mortality data from exposure of rainbow trout to test material for 96 hours are given in Table 1 (attached).
- The relationship between percentage mortality and concentration is shown in Figure 1 (attached).
- Environmental measurements (pH, temperatuire and oxygen concentration) are given in Appendix 1 (attached).
- Analysis of mortality data was conducted using the moving average method of Thompson (1947).
- The highest test concentration resulting in 0 % mortality was determined to be 1.0 mg/L.
- The lowest test concentration resulting in 100 % mortality was determined to be 3.2 mg/L.
- The No Observed Effect Concentration (NOEC) was determined to be 1.0 mg/L based on zero mortalities and the absence of any behavioural reactions to exposure at that concentration.
- The relationship between median lethal concentration and time is presented in Figure 2 (attached).

BEHAVIOURAL OBSERVATIONS
- Marked reactions to exposure (other than death) were lethargy, lying at the bottom of vessels, loss of equilibrium and the presence of moribund fish.
- Observations are summarised in Table 2 (attached).
Validity criteria fulfilled:
yes
Conclusions:
The LC50 (96 h) value was determined to be 1.7 mg/L. The highest test concentration resulting in 0 % mortality was determined to be 1.0 mg/L, the lowest test concentration resulting in 100 % mortality was determined to be 3.2 mg/L and the No Observed Effect Concentration (NOEC) was reported as 1.0 mg/L.
Executive summary:

In a study performed according to OECD 203 and EU Method C.1 (Handley et al, 1994) rainbow trout were exposed to five nominal concentrations of test material plus dechlorinated laboratory tap water control as the control for 96 hours under semi-static conditions. Ten fish were placed into the test chamber for each treatment group and the control group. Nominal concentrations of test item selected following a range-finding study were 1.0, 1.8, 3.2, 5.6 and 10 mg/L. Verification of test item concentration took place at 0, 24 and 96 hours using a spectrophotometric method. Chemical analysis showed the measured test concentrations to be near nominal and LC50 values were calculated in terms of nominal concentrations. No abnormalities were detected in the control group or the 1.0 mg/L test item group during the 96 hour test period. However, abnormalities were reported at other test concentrations and all fish in the 3.2, 5.6 and 10.0 mg/L test groups died during the study. The LC50 (96 h) value was determined to be 1.7 mg/L. The highest test concentration resulting in 0 % mortality was determined to be 1.0 mg/L, the lowest test concentration resulting in 100 % mortality was determined to be 3.2 mg/L and the No Observed Effect Concentration (NOEC) was reported as 1.0 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
21 April 1993 to 25 April 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
EPA OTS 797.1400 (Fish Acute Toxicity Test)
Version / remarks:
EPA/OTS guidelines for testing the environmental effects of chemicals on fish (U.S. EPA, 1985, 1987)
Deviations:
no
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
TEST DILUTION WATER
- The dilution water used during the performance of the acute toxicity test was reconstituted, deionised, well water.
- Representative samples of the dilution water were analysed for the presence of pesticides, PCBs and toxic metals (see Appendix IV, attached).
- In addition, representative samples of the dilution water source were analysed monthly for total organic carbon (TOC) concentration.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISMS
- Source: Wellington River Aquafarm, Plymouth, Massachusetts, USA.
- Prior to testing, the fish were held in a 500 L fibreglass tank under a photoperiod of 16 hours light and 8 hours darkness.
- The well water that flowed into the tank had a total hardness as CaCO3 of 25 to 27 mg/L, total alkalinity as CaCO3 of 21 to 23 mg/L and a specific conductivity range of 110 to 120 μmhos/cm.
- Other parameters monitored in the holding tank were pH (6.8-7.0), dissolved oxygen concentration (87-89 % saturation) and flow rate (10.6-12.5 tank volume replacements/day).
- Test fish were maintained under these conditions for a minimum of 14 days at a temperature of 12-13 °C.
- Fish were fed a dry commercial pelleted food ad libitum, daily, except during the 48 hours prior to testing. Representative samples of food were analysed for the presence of pesticides, PCBs and toxic metals. Food sources were considered to be of acceptable quality since the total concentration of pesticides measured was less than 0.3 mg/kg (ASTM, 1985).
- No mortality was observed among the test fish population during the 48-hour period prior to test initiation.
- A representative sample (N=30) of fish from the test population had a mean weight of 0.43 g (range 0.26 to 0.93 g) and a mean total length of 39 mm (range 32 to 45 mm).

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
- Total hardness as CaCO3 was 36 mg/L
- Total alkalinity as CaCO3 was 26 mg/L
Test temperature:
11 to 12 °C
pH:
6.9-7.5 (see Table 1, attached)
Dissolved oxygen:
8.0-9.8 mg/L (equivalent to 74-89 % saturation; see Table 1, attached)
Salinity:
Not applicable
Conductivity:
Specific conductivity 100 μmhos/cm
Nominal and measured concentrations:
PRELIMINARY TEST
- Nominal concentrations of 0.10, 10.0 and 100 mg A.I./L

DEFINITIVE TEST
- Nominal concentrations of 0.47, 0.79, 1.3, 2.3, 3.6, 6.0 and 10 mg A.I./L
Details on test conditions:
PRELIMINARY TEST
- A preliminary range-finding test was conducted in which rainbow trout were exposed under static conditions to nominal concentrations ranging from 0.10 to 100 mg A.I./L
- For the duration of the 96-hour exposure period, the 100 mg/L treatment level was observed to be a slightly cloudy solution.

DEFINITIVE TEST
- The static toxicity test was conducted in 18.9 L glass aquaria which contained 15 L of test solution.
- The exposure solution in each aquarium had a depth of 18.4 cm and a surface area of 819 cm2.
- During the study, seven test concentrations and a dilution water control were maintained in duplicate.
- Stock solution (20 mg A.I./mL) was prepared by diluting 2.7518 g (1.000 g as A.I.) of the test material in 50 mL of distilled water.
- Exposure solutions were then prepared by adding the appropriate amount of the 20 mg A.I./mL stock solution to 15 L of dilution water to give the desired test concentration.
- All exposure solutions were observed to be clear and colourless; they contained no visible sign of undissolved test material such as precipitate or surface film.
- One set of control aquaria was also established which contained the same dilution water and was maintained under the same conditions as the test aquaria but contained no test item.
- The test was initiated when rainbow trout were selected impartially from the holding tank and added to each test vessel (10 fish per replicate; 20 fish per treatment level).
- The resulting test organism loading concentration was 0.29 grams of biomass per litre of test solution.
- Fish were not fed during the study.
- Test vessels were positioned in stratified random order in a waterbath designed to maintain test solution temperatures at 12 ± 1 °C.
- Test solutions were not aerated.
- The photoperiod during testing was the same as that provided in the fish culture area.
- The test area was illuminated Durotest Vitalite and General Electric fluorescent light which provided light of an intensity of 70 footcandles at the surface of the solution.
- The test are received a regulated photoperiod of 16 hours light and 8 hours darkness. Sudden transitions from light to dark and vice versa were avoided.

TEST MONITORING
- All aquaria were examined at 0, 24, 48, 72 and 96 hours when mortalities were recorded, dead fish were removed and observations of the fish and of the physical characteristics of the test solutions were recorded
- At test initiation, and subsequently every 24 hours, the pH, dissolved oxygen concentration and temperature were measured in each replicate for all test concentrations and the control.
- Continuous temperature monitoring of the surrounding water in the waterbath was also performed.
- pH was measured using a LaMotte Model HA pH meter and a Jenco 601A pH meter with combination electrodes.
- Dissolved oxygen concentration was measured witha Yellow Springs Instrument (YSI) Model #67 dissolved oxygen meter and probe.
- Daily temperature was measured with a Fisher Scientific Min/Max thermometer.
- Light intensity was measured with a General Electric type 214 light meter.
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits
Remarks:
1.3-2.3 mg A.I./L
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits
Remarks:
1.3-2.3 mg A.I./L
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits
Remarks:
1.3-2.3 mg A.I./L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
TEST DILUTION WATER
- No pesticides, PCBs or toxic metals were detected at concentrations that were considered toxic in any of the water samples analysed. This finding was in agreement with U.S. EPA and ASTM guidelines (ASTM, 1989).
- Total organic carbon (TOC) concentration for the dilution water ranged from 0.82 to 1.5 mg/L during the months of October 1992 to March 1993. This result confirmed the acceptability of the dilution water for bioassays.

PRELIMINARY TEST
- Mortality of 100 % was observed for rainbow trout exposed to treatment levels of 10 and 100 mg A.I./L after 24 hours.
- No mortality was observed at test termination for rainbow trout exposed to 0.10 or 1.0 mg A.I./L. However, all of the surviving fish exposed to the 1.0 mg A.I./L test concentration exhibited sublethal effects such as loss of equilibrium.
- Based on these results, nominal concentrations of 0.47, 0.79, 1.3, 2.3, 3.6, 6.0 and 10 mg A.I./L were chosen for the definitive study.

DEFINITIVE TEST
- Results of the water quality parameters measured during the test are presented in Table 1 (attached).
- The pH, dissolved oxygen concentration and temperature were unaffected by the concentration of test item and remained within acceptable ranges for the survival of rainbow trout.
- Daily measurement of the temperature in the test solutions and continuous temperature monitoring of the surrounding water in the water bath established that the exposure solution temperature ranged from 11 to 12 °C throughout the definitive study.
- Nominal concentrations tested, the corresponding cumulative percent mortality and the observations made during the definitive exposure are presented in Table 2 (attached).
- Mortality of 100 % was observed among fish exposed to 3.6, 6.0 and 10 mg A.I./L after 24 hours exposure.
- No mortality or sublethal effects (e.g. loss of equilibrium) were observed at test termination for fish exposed to 0.47, 0.79 and 1.3 mg A.I./L test concentrations.
- Based on these data it was established that the effects observed were dose dependent.
- The 96-hour concentration-response (mortality) curve established for this study is presented in Figure 1 (attached).
Reported statistics and error estimates:
- Nominal concentrations tested and the corresponding biological-response data (mortality) derived from the toxicity test were used to estimate 24, 48, 72 and 96 hour median effect concentrations (LC50) and 95 % confidence intervals.
- The LC50 is defined as the concentration of the test material in dilution water which caused mortality of 50 % of the test organism population at the stated time interval.
- If at least one test concentration caused mortality of ≥ 50 % of the test population, a computer program modified from the program of C. Stephan (Peltier, 1985) was used to calculate the LC50 values and the 95 % confidence intervals.
- Three statistical methods were available in the computer program: moving average angle analysis, probit analysis and nonlinear interpolation with 95 % confidence intervals calculated by binomial probability.
- Moving average angle and probit analyses yield statistically sound results only if at least two concentrations produce mortality of between 0 and 100 % in the test population.
- The selection of reported LC50 values and 95 % confidence intervals was based upon an examination of the database and the results of the computer analysis.
- Selection criteria included the establishment of a concentration-effect relationship, the number of concentrations causing partial responses and the span of responses bracketing the LC50 value.
- If two or more statistical methods produced acceptable results, then the method which yielded the smallest 95 % confidence interval was selected.
- The No Observed Effect Concentration (NOEC) during the 96 hour exposure period was also determined. The NOEC is defined as the highest concentration tested and below which there were no toxicant-related mortality or physical and behavioural abnormalities (e.g. loss of equilibrium) with respect to the control.
Validity criteria fulfilled:
yes
Executive summary:

In a non-GLP study (Collins, 1993) rainbow trout were exposed to seven nominal concentrations of active ingredient plus reconstituted, deionised, well water as the control for 96 hours under static conditions. These systems were maintained in duplicate and ten fish were placed into the test vessel for each replicate (20 fish per treatment level and the control group). Based on the results of a preliminary study, nominal concentrations of 0.47, 0.79, 1.3, 2.3, 3.6, 6.0 and 10 mg a.i./L were chosen for the definitive study. Analytical monitoring of active ingredient concentration was not performed. Mortality of 100 % was observed among fish exposed to 3.6, 6.0 and 10 mg a.i./L after 24 hours exposure. No mortality or sublethal effects (e.g. loss of equilibrium) were observed at test termination for fish exposed to 0.47, 0.79 and 1.3 mg a.i./L test concentrations. Based on these data it was established that the effects observed were dose dependent and the LC50 (96 h) was reported as 1.7 mg a.i./L. The No Observed Effect Concentration (NOEC) was determined to be 1.3 mg a.i./L.

Description of key information

KEY STUDY

The 96 -h LC50 was determined to be 1.7 mg/L using Onchorhynchus mykiss. The highest test concentration resulting in 0 % mortality was determined to be 1.0 mg/L, the lowest test concentration resulting in 100 % mortality was determined to be 3.2 mg/L and the No Observed Effect Concentration (NOEC) was reported as 1.0 mg/L (OECD Guideline No. 203 referenced as Method C.1 of Commission Directive 92/69/EEC).

SUPPORTING STUDY

The 96 -h LC50 was reported as 1.7 mg a.i./L using Onchorhychus mykiss. The No Observed Effect Concentration (NOEC) was determined to be 1.3 mg a.i./L. (equivalent or similar to EPA OTS 797.1400 Fish Acute Toxicity Test).

Key value for chemical safety assessment

LC50 for freshwater fish:
1.7 mg/L

Additional information

In the key GLP study performed according to OECD 203 and EU Method C.1 (Handley et al, 1994) rainbow trout were exposed to five nominal concentrations of test material plus dechlorinated laboratory tap water control as the control for 96 hours under semi-static conditions. Ten fish were placed into the test chamber for each treatment group and the control group. Nominal concentrations of test item selected following a range-finding study were 1.0, 1.8, 3.2, 5.6 and 10 mg/L. Verification of test item concentration took place at 0, 24 and 96 hours using a spectrophotometric method. Chemical analysis showed the measured test concentrations to be near nominal and LC50 values were calculated in terms of nominal concentrations. No abnormalities were detected in the control group or the 1.0 mg/L test item group during the 96 hour test period. However, abnormalities were reported at other test concentrations and all fish in the 3.2, 5.6 and 10.0 mg/L test groups died during the study. The LC50 (96 h) value was determined to be 1.7 mg/L. The highest test concentration resulting in 0 % mortality was determined to be 1.0 mg/L, the lowest test concentration resulting in 100 % mortality was determined to be 3.2 mg/L and the No Observed Effect Concentration (NOEC) was reported as 1.0 mg/L.

In a non-GLP supporting study (Collins, 1993) rainbow trout were exposed to seven nominal concentrations of active ingredient plus reconstituted, deionised, well water as the control for 96 hours under static conditions. These systems were maintained in duplicate and ten fish were placed into the test vessel for each replicate (20 fish per treatment level and the control group). Based on the results of a preliminary study, nominal concentrations of 0.47, 0.79, 1.3, 2.3, 3.6, 6.0 and 10 mg a.i./L were chosen for the definitive study. Analytical monitoring of active ingredient concentration was not performed. Mortality of 100 % was observed among fish exposed to 3.6, 6.0 and 10 mg a.i./L after 24 hours exposure. No mortality or sublethal effects (e.g. loss of equilibrium) were observed at test termination for fish exposed to 0.47, 0.79 and 1.3 mg a.i./L test concentrations. Based on these data it was established that the effects observed were dose dependent and the LC50 (96 h) was reported as 1.7 mg a.i./L. The No Observed Effect Concentration (NOEC) was determined to be 1.3 mg a.i./L.