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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March to 31 March 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
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 and 48 hours.
Vehicle:
no
Details on test solutions:
TEST WATER
- Reconstituted water as defined in Appendix 3 (attached).
Test organisms (species):
other: Daphnia magna Straus
Details on test organisms:
TEST SPECIES
- Source: Laboratory culture originating from a strain supplied by the Institut National de Recherche Chimique Appliquee (I.R.CH.A.), France
- Culture: In polypropylene vessels containing 2 L of reconstituted water at 21 °C. Cultures were fed daily with a suspension of mixed algae (predominantly Chlorella spp.). Culture condiutions ensured that reproduction was by parthenogenesis.
- Selection: Gravid adults were isolated 24 hours prior to initiation of the test. Young daphnids produced overnight were used for testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Theoretical total hardness 270 mg/L as CaCO3
Test temperature:
21 °C
pH:
7.6 (see Appendix 1, attached)
Dissolved oxygen:
7.9-8.4 mg O2/L (see Appendix 1, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
PRELIMINARY STUDY
- Nominal test item concentrations of 0.10, 1.0, 10 and 100 mg/L.

DEFINITIVE STUDY
- Nominal test item concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L.
Details on test conditions:
METHOD OF PREPARATION
- Test material (500 mg) was dispersed in reconstituted water with the aid of ultrasonic disruption and the volume was adjusted to 5 L to give 100 mg/L.
- Dilutions were made from the 100 mg/L solution to give the test series.

PRELIMINARY STUDY
- Test item concentrations for the definitive test were assigned following a range-finding test involving exposure of 10 daphnids in four treatment groups.

EXPOSURE CONDITIONS FOR THE DEFINITIVE TEST
- Test vessels: Glass jars containing 250 mL test solution and covered to reduce evaporation.
- Experimental design: Nine test concentrations plus one control, each in duplicate (20 animals per concentration, 10 animals per replicate).
- Method of initiation: Daphnia were placed in the test solutions after addition of the test material.
- .Loading: 25 mL test solution per organism.
- Photoperiod: 16 h light and 8 h dark.
- Aeration: None.
- Environmental measurements: Temperature was recorded daily. Dissolved oxygen concentrations and pH were recorded at study initiation and termination.
- Criteria of effect: Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits
Remarks:
11-16 mg/L
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits
Remarks:
2.1-2.9
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
PRELIMINARY STUDY
- Immobilisation was observed at test item concentrations of 10 and 100 mg/L.
- No effect was seen when daphnids were exposed to 0.1 and 1.0 mg/L and these findings were used to indicate the choice of concentration for the definitive test.

DEFINITIVE STUDY
- Cumulative immobilisation data are given in Table 1 (attached).
- The relationship between percentage immobilisation and concentration at 24 and 48 hours are given in Figures 1 and 2 (attached).
- Environmental measurements (pH, temperature and oxygen concentration) are presented in Appendix 1 (attached).
- The No Observed Effect concentration (NOEC) was based upon zero immobilisation at the stated test concentration.

VERIFICATION OF TEST CONCENTRATION
- Chemical analysis of test solutions at 0 and 48 hours showed the measured test concentrations to be near nominal and it was considered justifiable to calculate EC50 results in terms of nominal test concentration only.
Reported statistics and error estimates:
- Analysis of immobility data was via the moving average method of Thompson (1947).
Validity criteria fulfilled:
yes
Conclusions:
The EC50 (48 h) value was determined to be 2.5 mg/L and the No Observed Effect Concentration (NOEC) was reported as 1.0 mg/L at 48 hours.
Executive summary:

In a study performed according to OECD 202 and EU Method C.2 (Handley et al, 1994) Daphnia magna were exposed in duplicate to nine nominal concentrations of test material plus reconstituted water as the control for 48 hours under static conditions (20 animals per concentration; 10 daphnids per replicate). Nominal concentrations of test item selected following a range-finding study were 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L. Verification of the control, 1.0, 3.2, 10, 32 and 100 mg/L test item concentrations took place at 0 and 48 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. The EC50 (48 h) value was determined to be 2.5 mg/L and the No Observed Effect Concentration (NOEC) was reported as 1.0 mg/L at 48 hours.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 March 1993 to 25 March 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: TSCA Test Standard 797-1300
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OTS 797.1300 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
EPA/OTS guidelines for testing the environmental effects of chemicals on daphnids (U.S. EPA, 1985, 1987)
Deviations:
no
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
TEST DILUTION WATER
- Culture water was prepared by fortifying well water based on the formula for hard water (ASTM, 1999) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants.
- 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):
Daphnia magna
Details on test organisms:
TEST ORGANISMS
- Source: Laboratory cultures maintained at Springbourn Laboratories, Inc.
- The daphnid culture area received a regulated photoperiod of 16 hours light and 8 hours darkness.
- Light at an intensity of 30-100 footcandles was provided by Durotest Vitalite and General Electric Coolwhite fluorescent bulbs at the surface of the solutions.
- Daphnids were fed a combination of 2.0 mL of Ankistrodesmus falcatus (approximately 4.0 x 10E07 cells/mL) and 0.5 mL of trout food suspension (5.0 mg/mL).
- Representative samples of each food source 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).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
- Total hardness as CaCO3 was 160-180 mg/L
- Total alkalinity as CaCO3 was 110-130 mg/L
Test temperature:
20
pH:
8.1-8.3 (see Table 1, attached)
Dissolved oxygen:
8.3-9.2 mg/L (equivalent to 94-97 % saturation; see Table 1, attached)
Salinity:
Not applicable
Conductivity:
400-600 µmhos/cm
Nominal and measured concentrations:
PRELIMINARY TEST
- Nominal concentrations of 0.1, 1.0, 10, 100 and 1000 mg A.I./L

DEFINITIVE TEST
- Nominal concentrations of 1.6, 2.6, 4.3, 7.2, 12 and 20 mg A.I./L
Details on test conditions:
PRELIMINARY TEST
- A preliminary range-finding test was conducted in which Daphnia magna were exposed under static con ditions to nominal concentrations ranging from 0.10 to 1000 mg A.I./L
- For the duration of the 96-hour exposure period, the two highest treatment levels were observed to be slightly cloudy.

DEFINITIVE TEST
- The toxicity test was conducted in 250 mL glass beakers which contained 200 mL of test solution.
- The exposure solution in each aquarium had a depth of 602 cm and a surface area of 33 cm2.
- Stock solution (10 mg A.I./mL) was prepared by diluting 0.2750 g (1.000 g as A.I.) of the test material in 10 mL of distilled water.
- The resulting stock solution was observed to be clear and colourless and contained no visible sign of undissolved test material.
- Exposure solutions were then prepared by adding the appropriate amount of the 10 mg A.I./mL stock solution to 1.0 L of dilution water to give the desired test concentration.
- Each exposure solution was mixed with a magnetic laboratory stirrer and Teflon coated stir bar for 30 seconds and was then divided into four replicate beakers (A, B, C and D) with 200 mL per replicate vessel.
- One set of control vessels was also established which contained the same dilution water and was maintained under the same conditions as the test vessels but did not contain test item.
- Test vessels were placed in stratified random order in a temperature controlled water bath designed to maintain test solution temperatures at 20 ± 2 °C.
- The test are was illuminated with Durotest Vitalite and General Electric Coolwhite fluorescent lights at an intensity of 80 footcandles at the solution surface.
- The photoperiod during testing was the same as that provided in the daphnid culture area. Sudden transitions from light to dark and vice versa were avoided.
- The definitive study was initiated when daphnids ≤ 24 hours old were selected impartially and distributed to each concentration and the control (five daphnids per vessel).
- Daphnids were not fed during exposure to test material.

TEST MONITORING
- The number of immobilised daphnids in each replicate test vessel was recorded at 24 and 48 hours.
- Biological observations and observations of the physical characteristics of the exposure solutions were also made and recorded at 0, 24 and 48 hours.
- The pH, dissolved oxygen concentration and temperature were measured for each treatment level (replicate A) and the control at 0, 24 and 48 hours.
- Total hardness, total alkalinity and specific conductance were measured at 0-hours in replicate E (dilution water control) and the highest treatment level solution (20 mg A.I./L nominal).
- The temperature of the surrounding water in the water bath was continuously monitored throughout the exposure period.
- Total hardness concentration was measured by the EDTA titrimetric method and total alkalinity concentration was determined by potentiometric titration to an endpoint of pH 4.5 (APHA et al, 1985).
- Specific conductivity was measured with a Yellow Springs Instrument Company (YSI) Model #33 salinity-conductivity-temperature meter and probe.
- The pH values were obtained using a Jenko Model 601A pH meter and combination electrode.
- Dissolved oxygen concentration was measured with a YSI Model #57 dissolved oxygen meter and probe.
- Daily temperature was measured with a Fisher Scientific alcohol thermometer.
- Continuous temperature monitoring was performed using a Fisher Scientific Min/Max thermometer.
- Light intensity was measured with a General Electric type 214 light meter.
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
7.7 g/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits
Remarks:
4.3-12 mg A.I./L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
4.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits
Remarks:
3.8-5.0 mg A.I./L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
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 ASTM Standard Practice (ASTM, 1989).
- The TOC concentration of the dilution water source was determined to be 1.5 mg/L for the month of March 1993. This result confirmed the suitability of the dilution water for bioassays.

PRELIMINARY TEST
- Total (100 %) immobilisation was observed among daphnids exposed to 100 and 1000 mg A.I./L treatment levels.
- After 48 hours of exposure, 80 % immobilisation was observed among daphnids exposed to the 10 mg A.I./L concentration and all of the mobile organisms were observed to be erratic.
- No immobilisation or sublethal effects (e.g. lethargy, erratic behaviour) were observed among daphnids exposed to the remaining concentrations tested (0.1 and 1.0 mg A.I./L).
- Based on these results, nominal concentrations of 1.6, 2.6, 4.3, 7.2, 12 and 20 mg A.I./L were chosen for the definitive study.

DEFINITIVE TEST
- Results of the water quality parameters measured during the definitive toxicity test are presented in Table 1 (attached).
- Analysis of the control solution and the 20 mg A.I./L test solution at test initiation established a total hardness (as CaCO3) of 160 and 170 mg/L respectively. The total alkalinity (as CaCO3) was established as 110mg/L and the specific conductivity of each solution was found to be 500 µmhos/cm. Based on these results it was determined that water quality parameters were not affected by test item concentration and remained within acceptable ranges for survival of daphnids.
- 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 19 to 20 °C throughout the definitive study.
- Nominal concentrations tested, the corresponding cumulative percent immobilisation and the observations made during the definitive exposure are presented in Table 2 (attached).
- After 24 hours exposure, 100 % immobilisation was observed among daphnids exposed to the two highest concentrations (12 and 20 mg A.I./L).
- At test termination (48 hours), immobilisation was observed among daphnids exposed to the 4.3 and 7.2 mg A.I./L treatment levels. The levels of immobilisation were 55 % and 90 % respectively. Immobilisation of 5 % was observed among daphnids exposed to the 2.6 mg A.I./L treatment level.
- Sublethal effects (e.g. lethargy, erratic behaviour) were observed among several of the mobile daphnids exposed to the 4.3 mg A.I./L treatment level.
- No immobilisation or sublethal effects were observed among daphnids exposed to the remaining concentration tested (1.6 mg A.I./L).
- Based on these data, it was established that the effects observed during the study were clearly concentration dependent.
- The 48-hour concentration-repsonse (immobilisation) curve for the study is presented in Figure 1 (attached). The slope of the line was calculated to be 6.6579.
Reported statistics and error estimates:
- Nominal concentrations tested and the corresponding biological-response data (immobilisation) derived from the toxicity test were used to estimate 24 and 48 hour median effect concentrations (EC50) and 95 % confidence intervals.
- The EC50 is defined as the concentration of the test material in dilution water which caused immobilisation of 50 % of the test organism population at the stated time interval.
- If at least one test concentration caused immobilisation 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, probi t 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 con centrations produce mortality of between 0 and 100 % in the test population.
- The selection of reported EC50 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 conce ntrations causing partial responses and the span of responses bracketing the EC50 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 determ ined. The NOEC is defined as the highest concentration tested and below which there were no toxicantrelated mortality or physical and behavioural abnormalities (e.g. loss of equilibrium) with respect to the control.
Validity criteria fulfilled:
yes
Conclusions:
Based on nominal concentrations of test item, the 48 hour EC50 value was calculated by probit analysis to be 4.3 mg A.I./L (95 % confidence interval 3.8-5.0 mg A.I./L). The No Observed Effect Concentration established for the study was determined to be 1.6 mg A.I./L.
Executive summary:

In a non-GLP study (Collins, 1993) Daphnia magna were exposed to seven nominal concentrations of active ingredient plus fortified well water as the control for 48 hours under static conditions. Five replicates were established for each test concentration and the control. Five daphnids were placed into each vessel (four replicates containing test item plus the control vessel). Based on the results of a preliminary study, nominal concentrations of 1.6, 2.6, 4.3, 7.2, 12 and 20 mg A.I./L were chosen for the definitive study. Analytical monitoring of active ingredient concentration was not performed. After 24 hours exposure, 100 % immobilisation was observed among daphnids exposed to the two highest concentrations (12 and 20 mg A.I./L). At test termination (48 hours), immobilisation was observed among daphnids exposed to the 4.3 and 7.2 mg A.I./L treatment levels. The levels of immobilisation were 55 % and 90 % respectively. Immobilisation of 5 % was observed among daphnids exposed to the 2.6 mg A.I./L treatment level. Sublethal effects (e.g. lethargy, erratic behaviour) were observed among several of the mobile daphnids exposed to the 4.3 mg A.I./L treatment level. No immobilisation or sublethal effects were observed among daphnids exposed to the remaining concentration tested (1.6 mg A.I./L). Based on these data it was established that the effects observed were dose dependent and the EC50 (48 h) was reported as 4.3 mg A.I./L. The No Observed Effect Concentration (NOEC) was determined to be 1.6 mg A.I./L.

Description of key information

KEY STUDY

The EC50 (48 h) value (Daphnia magna Strauss) was determined to be 2.5 mg/L and the No Observed Effect Concentration (NOEC) was reported as 1.0 mg/L at 48 hours (OECD Guideline No. 202 referenced as Method C.2 of Commission Directive 92/69/EEC).

SUPPORTING STUDY

EC50 (48 h) was reported as 4.3 mg A.I./L (Daphnia magna). The No Observed Effect Concentration (NOEC) was determined to be 1.6 mg A.I./L (TSCA Test Standard 797 -1300; equivalent or similar to EPA OTS 797.1300 Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.5 mg/L

Additional information

In the key GLP study performed according to OECD 202 and EU Method C.2 (Handley et al, 1994) Daphnia magna were exposed in duplicate to nine nominal concentrations of test material plus reconstituted water as the control for 48 hours under static conditions (20 animals per concentration; 10 daphnids per replicate). Nominal concentrations of test item selected following a range-finding study were 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L. Verification of the control, 1.0, 3.2, 10, 32 and 100 mg/L test item concentrations took place at 0 and 48 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. The EC50 (48 h) value was determined to be 2.5 mg/L and the No Observed Effect Concentration (NOEC) was reported as 1.0 mg/L at 48 hours.

In a non-GLP supporting study (Collins, 1993) Daphnia magna were exposed to seven nominal concentrations of active ingredient plus fortified well water as the control for 48 hours under static conditions. Five replicates were established for each test concentration and the control. Five daphnids were placed into each vessel (four replicates containing test item plus the control vessel). Based on the results of a preliminary study, nominal concentrations of 1.6, 2.6, 4.3, 7.2, 12 and 20 mg a,i./L were chosen for the definitive study. Analytical monitoring of active ingredient concentration was not performed. After 24 hours exposure, 100 % immobilisation was observed among daphnids exposed to the two highest concentrations (12 and 20 mg a.i./L). At test termination (48 hours), immobilisation was observed among daphnids exposed to the 4.3 and 7.2 mg a.i./L treatment levels. The levels of immobilisation were 55 % and 90 % respectively. Immobilisation of 5 % was observed among daphnids exposed to the 2.6 mg a.i./L treatment level. Sublethal effects (e.g. lethargy, erratic behaviour) were observed among several of the mobile daphnids exposed to the 4.3 mg a.i./L treatment level. No immobilisation or sublethal effects were observed among daphnids exposed to the remaining concentration tested (1.6 mg a.i./L). Based on these data it was established that the effects observed were dose dependent and the EC50 (48 h) was reported as 4.3 mg a.i./L. The No Observed Effect Concentration (NOEC) was determined to be 1.6 mg a.i./L.