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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:
key study
Study period:
21 - 26 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992 (updated 2009)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
Commission Regulation (EC) No. 440/2008
Deviations:
no
Principles of method if other than guideline:
In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA (Guidance on the Application of the CLP Criteria, Version 5.0 – July 2017)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at ambient temperature in the dark.
- Stability under test conditions: Stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72 and 96 hours.
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A nominal amount of test item (1100 mg) was dissolved in 22 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters used to pre-condition the filter was discarded) to give the nominal 50 mg/L test concentration.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : See above

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS: N/A
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 50 mg/L (nominal)
- Sampling method: Water samples were taken from the control and test group, from fresh and old media throughout the 96-Hour test period, for quantitative analysis. The samples at 0, 24 (old media) and 72 hours (fresh media) and 96 hours (old media) were taken for analysis immediately
- Sample storage conditions before analysis: Samples from freshly prepared media at 24 and 48 hours and from aged media at 48 and 72 hours were taken and stored frozen
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (1100 mg) was dissolved in 22 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters used to pre-condition the filter was discarded) to give the 50 mg/L test concentration.
- Eluate: The test water used for the initial experiment and definitive test was the same as that used to maintain the stock fish. Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: N/A
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 20 July 2018.
- Age at study initiation (mean and range, SD): "Juvenile" - exact age not reported
- Length at study initiation (length definition, mean, range and SD): 5.5 cm (sd = 0.4)
- Weight at study initiation (mean and range, SD): 1.2 g (sd = 0.3)
- Method of breeding: N/A
- Maintenance of the brood fish: N/A
- Loading: 0.42 g bodyweight/L

ACCLIMATION
- Acclimation period: Fish were acclimatized to test conditions from 15 October 2018 to 22 October 2018.
- Acclimation conditions (same as test or not): Yes
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets which was discontinued approximately 25 hours prior to the start of the definitive test.
- Feeding frequency during acclimation: Not reported
- Health during acclimation (any mortality observed): There were three mortalities (<2%) in the 7 days prior to the start of the test.

QUARANTINE (wild caught)
- Duration: N/A
- Health/mortality: N/A

FEEDING DURING TEST
- Food type: No feeding during the test.
- Amount: N/A
- Frequency: N/A
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
As prescribed in test guideline.
Hardness:
140 mg/L as CaCO3
Test temperature:
14-15 ºC
pH:
7.5 - 8.2
Dissolved oxygen:
9.1 - 10.7 mg/L
Salinity:
N/A
Conductivity:
345 µS/cm at 20 ºC
Nominal and measured concentrations:
Nominal = 50 mg/L
Mean measured = 47 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Fish tank
- Type (delete if not applicable): Open (covered to reduce evaporation).
- Material, size, headspace, fill volume: Glass, 25-30 L capacity filled to 20 L
- Aeration: Yes, via narrow bore glass tube.
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A, semi static test.
- Renewal rate of test solution (frequency/flow rate): Daily renewals.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: 0.42 g bodyweight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Al: 7.46 mg/L; Fe: 19.9 µg/L; Mn: 2.9 µg/L; Na: 22.35 mg/L; Cu: 0.02 mg/L; Pb: 0.51 µg/L; Ni: 3.1 µg/L
- Pesticides: 0 µg/L
- Chlorine: 0.32 mg/L
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and sub-lethal effects at 1, 3, 6, 25, 48, 72 and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A
- Justification for using less concentrations than requested by guideline: Limit test
- Range finding study
- Test concentrations: N/A
- Results used to determine the conditions for the definitive study: In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC50 value from either the Algal Growth Inhibition study (Envigo study number KH20LT) or Acute Toxicity to Daphnia magna study (Envigo study number HC56FR) is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The EC50 value obtained from the Algal Growth Inhibition study was the lowest of these two EC50 values, which was calculated to be 30 mg/L. Analytical results obtained from the Daphnia test showed measured concentrations were as low as 70% of nominal concentrations and hence the test was conducted at a single concentration of 50 mg/L in order to account for these potential differences.
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 47 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: Sub-lethal effects of exposure were observed at the test concentration. One fish was swimming at the bottom of the vessel one hour after exposure. One or two fish were swimming at the bottom of the vessel with their heads up 6 hours after exposure and subsequent observations.
- Observations on body length and weight:
- Other biological observations: Increased pigmentation was observed in six fish 96 hours after exposure.
- Mortality of control: 0
- Other adverse effects control: No
- Abnormal responses: None, other than those stated above.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
N/A

Table 1       Cumulative mortality data

 

Measured concentration

(mg/L)

Cumulative mortality (initial population = 7)

% Mortality

1 h

3 h

6 h

25 h

48 h

72 h

96 h

96 h

Control

0

0

0

0

0

0

0

0

47

0

0

0

0

0

0

0

0

 

Table 2       Sub-lethal effects

 

Measured concentration

(mg/L)

Sub-lethal effects

Time (h)

1

3

6

24

48

72

96

Control

None

6

7

7

7

7

7

7

Swimming at bottom of vessel

1

0

0

0

0

0

0

47

None

6

7

6

5

5

5

0

Swimming at bottom of vessel

1

0

0

0

0

0

0

Swimming at bottom of vessel with head up

0

0

1

2

2

2

1

Increased pigmentation

0

0

0

0

0

0

6

 

Table 3       Water quality parameters

 

Measured concentration

(mg/L)

Time (hours)

0 h (fresh media)

24 h (old media)

pH

Mg O2/L

Temperature ºC

pH

Mg O2/L

Temperature ºC

Control

7.8

10.6

14

8.1

10.1

14

47

8.1

10.5

14

8.2

10.0

15

 

Measured concentration

(mg/L)

Time (hours)

24 h (fresh media)

48 h (old media)

pH

Mg O2/L

Temperature ºC

pH

Mg O2/L

Temperature ºC

Control

7.6

10.6

14

7.6

9.3

14

47

8.0

10.7

14

7.9

10.1

14

 

Measured concentration

(mg/L)

Time (hours)

48 h (fresh media)

72h (old media)

pH

Mg O2/L

Temperature ºC

pH

Mg O2/L

Temperature ºC

Control

7.5

10.6

14

7.6

10.0

14

47

7.7

10.5

14

7.7

9.5

14

 

Measured concentration

(mg/L)

Time (hours)

72 h (fresh media)

96 h (old media)

pH

Mg O2/L

Temperature ºC

pH

Mg O2/L

Temperature ºC

Control

7.7

10.3

14

8.1

9.8

14

47

7.9

10.4

14

8.0

9.1

14

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated using the threshold approach and gave a 96-hour LC50 value of greater than 47 mg/L based on the mean measured concentration. The NOEC based on mortality was 47 mg/L.
Executive summary:

OECD 203 (2019) - In a 96-h acute toxicity study, rainbow trout (Oncorhynchus mykiss) were exposed to the test item at a mean measured concentration of 47 mg/L under semi-static conditions, with 24 h renewals. 

The test item solutions were prepared by stirring (50 mg/L nominal) test item in reconstituted freshwater (test media) using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to produce the nominal 50 mg/L test item solution. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72, and 96 h.

 

Single test vessel and control vessel (reconstituted freshwater only) replicates were each prepared containing 20 L of test solution and 7 fish (biomass loading = 0.42 g bw/L). Test vessel were covered to reduce evaporation and maintained at 14-15 ºC for 96 h with intermittent light dark cycles (16:8, respectively), during which time the fish were not fed.

Chemical analysis of the freshly prepared 0 h and 72 h test solutions showed measured concentrations of 46 and 48 mg/L, respectively. Similar concentrations (44 and 51 mg/L) were observed in the expired 24 and 96 h solutions, indicating that the test item was stable over the 24 h renewal period. The overall mean (arithmetic) measured concentration was 47 mg/L.

 

No mortality was observed in the test or control groups during the test. Sub-lethal effects of exposure were observed in the test group. One fish was swimming at the bottom of the vessel one hour after exposure. One or two fish were swimming at the bottom of the vessel with their heads up 6 hours after exposure and subsequent observations. Increased pigmentation was observed in six fish 96 hours after exposure.

 

Based on mortality, the 96 -h LC50 and NOEC were > 47 mg/L.

 

This toxicity study is classified as acceptable and satisfies the guideline requirement for acute toxicity in fish.

Description of key information

96 h LC50 = >47 mg/L; OECD 203; Squance, D. (2019)

Key value for chemical safety assessment

Additional information

OECD 203 (2019) - In a 96-h acute toxicity study, rainbow trout (Oncorhynchus mykiss) were exposed tothe test itemat a mean measured concentration of 47 mg/L under semi-static conditions, with 24 h renewals. 

The test item solutions were prepared by stirring (50 mg/L nominal) test item in reconstituted freshwater (test media) using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to produce the nominal 50 mg/L test item solution. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72, and 96 h.

 

Single test vessel and control vessel (reconstituted freshwater only) replicates were each prepared containing 20 L of test solution and 7 fish (biomass loading = 0.42 g bw/L). Test vessel were covered to reduce evaporation and maintained at 14-15 ºC for 96 h with intermittent light dark cycles (16:8, respectively), during which time the fish were not fed.

Chemical analysis of the freshly prepared 0 h and 72 h test solutions showed measured concentrations of 46 and 48 mg/L, respectively. Similar concentrations (44 and 51 mg/L) were observed in the expired 24 and 96 h solutions, indicating that the test item was stable over the 24 h renewal period. The overall mean (arithmetic) measured concentration was 47 mg/L.

 

No mortality was observed in the test or control groups during the test. Sub-lethal effects of exposure were observed in the test group. One fish was swimming at the bottom of the vessel one hour after exposure. One or two fish were swimming at the bottom of the vessel with their heads up 6 hours after exposure and subsequent observations. Increased pigmentation was observed in six fish 96 hours after exposure.

 

Based on mortality, the 96 -h LC50and NOEC were > 47 mg/L.

 

This toxicity study is classified as acceptable and satisfies the guideline requirement for acute toxicity in fish.