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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 September 1993 to 2 October 1993
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:
equivalent or similar to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 797.1400 (Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All tested concentrations
- Sampling method: Appropriate amounts of the test material were weighed onto individual microscope slides to prepare spike solutions. The test material was rinsed from the slides into a 2000 mg/L glass beaker using 1800 mL of hard blended water. A Teflon coated stir bar was added to each beaker. The beakers were placed on VWR Dylastirs, and the contents were stirred at a moderate rate for 90 minutes at room temperature. A 1500 mL subsurface water sample was collected from each spike beaker using a 100 mL glass pipet. Samples were collected from the test chambers in the same manner, 750 mL of water were removed from each replicate. The replicate samples were combined for a 1500 mL total sample. The 1500 mL water samples were then transferred to individual 2000 mL separatory funnels.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test concentrations were prepared by transferring appropriate weight of the test compound to the test chambers. The test material was weighed onto glass microscope slides. The test jar was filled with 15 L of hard blended water. A plastic squirt bottle was filled from the water in the jar. The test material on the slide was washed off the slide into the jar using the water in the squirt bottle. Any water left in the squirt bottle after all material was washed off the slide was also added to the jar. This process was repeated for all test chambers. A stir bar was added to the jars and the solutions were stirred for approximately 1.5 hours. All test concentrations were based on the total compound and not corrected for sample purity.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Source: in-house culture
- Length at study initiation (length definition, mean, range and SD): Total length 25 ± 2 mm and standard length of 20 ± 2 mm
- Weight at study initiation (mean and range, SD): 0.09 ± 0.03 g (wet weight)
- Maintenance of the brood fish: The fish were reared and maintained in hard blended water and were fed newly hatched brine shrimp, aquatic invertebrates, and/or commercially available fish food daily.

ACCLIMATION
- Acclimation period: The fish were acclimated to test temperature for 72 hours prior to testing.
- Acclimation conditions (same as test or not): The same as the test
- Type and amount of food during acclimation: Fish were not fed during the acclimation period
- Health during acclimation (any mortality observed): Fish were in good condition for testing
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
144-154 mg/L as CaCO3 (total hardness)
Test temperature:
22 ± 1 °C
pH:
7.6-8.3
Dissolved oxygen:
7.4-8.7 mg/L (dissolved oxygen concentration) 89-104 % saturation
Conductivity:
340-400 µmhos/cm
Nominal and measured concentrations:
0, 0.72, 1.3, 2.4, 4.4, 8.0 mg/L (nominal)
0, 0.46, 0.92, 1.4, 3.1, 7.5 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass vessels
- Material, size, headspace, fill volume: 5 gallon (19 L), glass vessels filled to 15 (equivalent to a depth of 29.9 cm).
- Aeration: No aeration
- Renewal rate of test solution (frequency/flow rate): Replaced after 24 hours (fish were transferred to new test solutions)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 0.06 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Hard blended dilution water prepared by blending naturally hard well water with reverse osmosis demineralised well water.
- Total organic carbon: < 1.0 mg/L
- Particulate matter: 6.9 mg/L
- Metals:
Aluminium <0.20 mg/L
Arsenic <0.010 mg/L
Cadmium <0.50 mg/L
Chromium < 0.0010 mg/L
Cobalt < 0.050 mg/L
Copper < 0.020 mg/L
Iron < 0.10 mg/L
Lead < 0.003 mg/L
Mercury < 0.20 mg/L
Nickel < 0.050 mg/L
Silver < 0.50 mg/L
Selenium < 0.005 mg/L
Zinc < 0.040 mg/L
- Alkalinity: 144-154 mg/L as CaCO3
- Culture medium different from test medium: No
- Intervals of water quality measurement: Monthly and biyearly

OTHER TEST CONDITIONS
- Photoperiod: 16 hour daylight period with 30 minute transition periods.
- Light intensity: 45-57 footcandles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : All organisms were observed every 24 hours for mortality and abnormal (sublethal effects). Dead individuals were removed from the test solutions.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: The first preliminary range finding test was conducted with concentrations of 0.5, 5.0, 20 and 50 mg/L. These were prepared using dimethylformamide as a solvent. Additional range finding studies were conducted without solvent.
The second and third range finding tests were conducted at the test concentrations of 1.0, 10, 100 and 40 mg/L, respectively.
- Results used to determine the conditions for the definitive study: The 10, 40 and 100 mg/L test concentrations caused 100 % mortality within 24 hours. The fish in the 1.0 mg/L solution were normal at 24 hours. A final test was conducted at 0.5 and 5.0 mg/L. The 5.0 mg/L solution resulted in 100 % mortality within 24 hours. The fish in the 0.5 mg/L solution were normal throughout the 96-hour test. A new 0.5 mg/L solution was prepared at 48 hours and the fish were transferred from the old to the new solution.
Based on the results of the preliminary test, five concentration of the test material, ranging in a geometric series from 0.72 to 8.0 mg/L, were selected for the definitive bioassay.
Reference substance (positive control):
no
Key result
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
0.79 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
1.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
0.97 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
< 0.46 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
< 0.72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: The 96 hour no observed effect concentration was not determined but was shown to be < 0.46 mg/L (measured value) or < 0.72 mg/L (nominal value), the lowest concentration tested. The abnormal effects of mortality, loss of equilibrium, fish on the bottom of the test chamber, and or quiescence were observed in all test concentrations except for the control during the 96 hour exposure period. The 0.72 mg/L (nominal) replicate B concentration had one dead fish at 96 hours. All other fish at this concentration were found to be normal throughout the study. New solutions were prepared every 24 hours and the fish were transferred from the 24 hour old solutions to the new solutions.
- Mortality of control: One control replicate A fish was lost during the transfer at 24 hours.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The analytical method was validated prior to the initiation of the definitive study. Fortification samples were prepared ranging from 1.1 to 20.9 mg/L. Recoveries ranged from 70.2 to 96 % over this concentration range.
The nominal test concentrations for this study were 0.72, 1.3, 2.4, 4.4, and 8.0 mg/L. Samples for analytical confirmation were collected at 0 (new) and 24 (old) hours, just prior to the test solution renewal. The measured test concentration at 0 hours were 0.563, 1.08, 1.75, 3.70 and 8.13 mg/L. These measured test concentrations ranged from 73 to 100 % of the nominal test concentrations. The measured test concentrations from the 24 hour old solutions were 0.356, 0.758, 1.05, 2.44 and 6.92 mg/L This decrease in measured values indicated that the test solutions were not stable under static test conditions. The percent loss between 0 and 24 hours was 37, 30, 40, 34 and 15 % for levels 1-5 respectively. The mean measured test concentrations for this study were 0.46, 0.92, 1.4, 3.1 and 7.5 mg/L. These values corresponded to 58-94 % of the nominal test concentrations. Nominal concentrations of the quality control samples prepared at each sampling point were 0.5, 2.0 and 10.0 mg/L. Recoveries of the quality control fortification for the study ranged from 59 to 103 % of the nominal fortification concentrations. Results for the 0 hour mid spike and for the low, mid and high spikes with the 24 hour test samples were confirmed by reanalysing the samples by HPLC. The results from the 24 hour fortification samples indicated a preparation error for the three samples.
Reported statistics and error estimates:
Calculations of the test material concentrations were performed using external standard analysis were performed using the VAX MULTICHROM Software.
Concentrations (µg/mL) of residues were determined directly from the standard curve using the following equation:
((µg/mL test material equivalents from standard curve equation)*(volume for analysis mL) * (dilution factor))/(volume extracted in mL)

The dilution factor compensates for the aliquot of sample extract taken for the derivatisation factor equals prederivatisation volume divided by the aliquot volume collected for derivatisation.
Statistical analysis of the concentration versus effect data was obtained by using an LC50 program. This calculated the LC50 on its 95 % confidence limits using the binomial, moving average, and probit tests. Three different methods of analysing the data were used since no one method of analysis is appropriate for all possible sets of data. If no mortality occurred or if a dose response could not be demonstrated over a reasonable range (<37 to > 63 %) and LC50 and/or its 95 % confidence limits could not be calculated. If the LC50 was calculated by more than one method, the method of calculation selected is the one which gave the narrowest range for the confidence limits. The dose response slope was calculated by transforming percent mortality to probit values using linear regression.

Table 1: Results of the definitive test

Nominal Concentration
(mg/L)

Mean Measured Concentration
(mg/L)

No. Organisms

24 hours

48 hours

72 hours

96 hours

Mortality

Observations

Cumulative Mortality

Observations

Cumulative Mortality

Observations

Cumulative Mortality

Observations

Control A

Control A

10

0

10N

0

9N*

0

9N

0

9N

Control B

Control B

10

0

10N

0

10N

0

10N

0

10N

0.72 A

0.46A

10

0

10N

0

10N

0

10N

0

10N

0.72 B

0.46B

10

0

10N

0

10N

0

10N

1

9N

1.3 A

0.92A

10

1

1 LOE; 8N

7

3 LOE/OB/Q

10

-

10

-

1.3 B

0.92B

10

1

1 LOE; 8N

7

1 Q; 2 LOE/OB/Q

10

-

10

-

2.4A

1.4A

10

10

-

10

-

10

-

10

-

2.4B

1.4B

10

10

-

10

-

10

-

10

-

4.4A

3.1A

10

10

-

10

-

10

-

10

-

4.4B

3.1B

10

10

-

10

-

10

-

10

-

8.0A

7.5A

10

10

-

10

-

10

-

10

-

8.0B

7.5B

10

10

-

10

-

10

-

10

-

* One fish lost during test solution renewal
N = Normal
LOE = Loss of equilibrium
Q = Quiescent
OB = Fish on the bottom of the test vessel

 

Validity criteria fulfilled:
yes
Conclusions:
The nominal duplicate test concentrations for this study were 0.72, 1.3, 2.4, 4.4 and 8.0 mg/L. Analysis of the test solutions at 0 and 24 hours yielded a recovery range of 73 to 100 % at 0 hours and 44 to 87 % at 24 hours. The test material did not appear stable under static test conditions based on this decrease in recovery between 0 and 24 hours. The test solutions were renewed every 24 hours. The mean measured concentrations were 0.46, 0.92, 1.4, 3.1 and 7.5 mg/L, respectively. The 96 hour LC50 for fathead minnow was determined to be 0.62 mg/L with confidence limits of 0.72 and 1.3 mg/L (nominal). A 96 hour no observed effect concentration was not determined, but was estimated to be < 0.46 mg/L (measured) or < 0.72 mg/L (nominal).
Executive summary:

The acute toxicity of the test material to fathead minnows was determined in a test conducted in accordance with OECD 203 and US EPA TSCA 40 CFR Part Guideline 797.1400. A preliminary range finder was performed using DMF as a solvent. The second and third range finding studies and the definitive test was performed without the use of a solvent.

The nominal duplicate test concentrations for this study were 0.72, 1.3, 2.4, 4.4 and 8.0 mg/L. Analysis of the test solutions at 0 and 24 hours yielded a recovery range of 73 to 100 % at 0 hours and 44 to 87 % at 24 hours. The test material did not appear stable under static test conditions based on this decrease in recovery between 0 and 24 hours. The test solutions were renewed every 24 hours. The mean measured concentrations were 0.46, 0.92, 1.4, 3.1 and 7.5 mg/L, respectively. The 96 hour LC50 for fathead minnow was determined to be 0.62 mg/L with confidence limits of 0.72 and 1.3 mg/L (nominal). A 96 hour no observed effect concentration was not determined, but was estimated to be < 0.46 mg/L (measured) or < 0.72 mg/L (nominal).

Description of key information

A study was performed to assess the acute toxicity of the test material to rainbow trout (Onchorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No. 203, “Fish Acute Toxicity Test” referenced as Method C.1 of Commission Directive 92/69/EEC, as well as US CFR Title 40 (part 797 Section 1400) and US EPA Draft Ecological Effects Test Guideline OPPTS 850.1075. Following preliminary range finding studies, fish were exposed in groups of ten to water accommodated fractions (WAFs) of the test material prepared using a method employing a single phase separation over a range of nominal loading rates of 0.32, 0.56, 1.0, 1.8, and 3.2 mg/L for a period of 96 hours under static test conditions. The number of mortalities and any sublethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the study until termination after 96 hours.

The 96 hour LL50 based on nominal loading rates was 1.4 mg/L loading rate WAF with 95% confidence limits of 1.3 to 1.6 mg/L. The No Observed Effect Loading rate was 0.56 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.4 mg/L

Additional information