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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:
19th January - 2nd February 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
Fish with an average length bigger than 3.0 +/- 1.0cm were introduced in the limit test (3.7 +/- 0.4cm). However, it is concluded this small deviation will not have had an impact on the results of the study.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and 100 mg/l
- Sampling method: 4.0 mL samples were taken at 0 h and 96 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was dissolved in the medium with a 15-16 minute stir time to create the highest test concentration of 100 mg/l.
- Differential loading: no, lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: test medium (adjusted ISO medium with a hardness of 180 mg CaCO3 per litre)
- Test concentration separation factor:
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions were clear and colourless at the end of the preparation procedure
- Other relevant information:
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Strain: Not reported
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): 3.7 +/- 0.4 cm
- Weight at study initiation (mean and range, SD): 0.68 +/- 0.28g
- Method of breeding: Not reported, but fish were F1 from a single parent-pair bred in UV treated water
- Maintenance of the brood fish: not reported

ACCLIMATION
- Acclimation period: at least 12 days after delivery
- Acclimation conditions (same as test or not): adjusted ISO medium and in optimum water quality parameters for the respective fish species, different to the test
- Type and amount of food during acclimation: pelleted fish food (Essence (300-500 µm), Coppens International bv, Helmond, The Netherlands). Amount not reported
- Feeding frequency during acclimation: daily with pelleted fish food
- Health during acclimation (any mortality observed): mortality during the seven days prior to the start of the test was less than 5%


FEEDING DURING TEST - No feeding from 24 hours prior to the test and during the total test period.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
180 mg CaCO3 per litre
Test temperature:
21-22 degrees C
pH:
7.7-7.9
Dissolved oxygen:
7.1-8.9 mg/L. Before the addition of the aeration at 2 days after exposure, DO decreased to 5.9 mg/L in the control group and 6.9 mg/L in the 100 mg/L test concentration.
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: control and 100 mg/L
Measured: control and 100 mg/L (100% of nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 10 L all glass vessels containing 9 L of test solution
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: glass, 10 L total volume with 1 L headspace
- Aeration: aeration was introduced after 2 days of exposure for approximately 6.25 hours
- No. of organisms per vessel: 7 per test group
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.53 g fish/litre

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium with adjusted hardness of CaCO3 per litre. Further details not reported
- Intervals of water quality measurement: dissolved oxygen, pH and temperature measured daily

OTHER TEST CONDITIONS
- Adjustment of pH: pH of test media adjusted to pH 7.7 +/- 0.3
- Photoperiod: 16 hours light : 8 hours dark
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and evidence of distressed fish was measured at 3.5, 24, 48, 72 and 96 hours following the start of the exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test with the control and 100 mg/L
- Justification for using less concentrations than requested by guideline: not applicable
RANGE FINDING STUDY
- Test concentrations: 0,0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no mortality observed, therefore a limit test using the maximum test concentration of 100 mg/L was used for the definitive exposure.
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol was tested at concentrations of 0.10, 0.22 and 0.46 mg/L in ISO-medium.
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: concluded by reviewer
Details on results:
- Other abnormalities: No clinical effects were observed in the control or test concentration during the exposure period
- Observations on body length and weight: Not measured
- Other biological observations: None reported
- Mortality of control: 0% mortality in the control
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: none reported
Results with reference substance (positive control):
100% mortality was observed in the highest concentration. LC50 = 0.32 mg/L
Reported statistics and error estimates:
Statistical analysis was not conducted because no mortality was observed in the control or test concentration.
Sublethal observations / clinical signs:

Table 1. Cumulative mortality of fish throughout the test duration

Nominal concentration (mg/l)

Initial number of fish

Cumulative mortality

Total mortality

3.5 hours

24 hours

48 hours

72 hours

96 hours

Control

7

0

0

0

0

0

0

100

7

0

0

0

0

0

0

Table 2. pH, dissolved oxygen (DO) and temperature in test vessels throughout the test duration

Nominal concentration (mg/L)

Day 0

Day 1

Day 2

Day 3

Day 4

pH

DO (mg/L)

Temperature

°C

pH

DO (mg/L)

Temperature

°C

pH

DO (mg/L)

Temperature

°C

pH

DO (mg/L)

Temperature

°C

pH

DO (mg/L)

Temperature

°C

Control

7.9

8.5

21

7.9

7.1

22

7.7

5.9*

21

7.7

7.2

21

7.9

7.4

21

100

7.9

8.9

21

7.9

8.6

22

7.7

6.9*

22

7.8

7.9

21

7.8

8.4

21

* Values display the dissolved oxygen prior to aeration. After aeration was added on this day, both values increased to 8.5 mg/L

Table 3. Nominal and measured concentrations of the test item at 0 and 96 hours

Nominal concentration (mg/l)

Measured concentration (mg/l)

Relative to nominal (%)

Relative to initial (%)

0 hours

96 hours

Control

Not detected

Not detected

Not applicable

Not applicable

100

100

99.8

100

100

Validity criteria fulfilled:
yes
Remarks:
No mortality was observed in the control group, test conditions remained constant, dissolved oxygen remained above 5 mg/L and the concentration of the test item was 100% of the nominal value.
Conclusions:
A 96-hour LC50 of >100 mg/L was determined for the effects of the test substance on Cyprinus carpio, based on measured concentrations. The study was conducted according to OECD test guideline 203, in accordance with GLP and the study met the validity criteria. However, due to a lack of detail reported in the analytical method, the study has been assigned a reliability score of 2.
Executive summary:

Charles River Laboratories (2018) is a GLP-compliant study following OECD guideline 203. A range finding test was conducted with the concentrations of 0.10, 1.0, 10 and 100 mg/L. There was no mortality observed in the control or test concentrations at after 96 hours. Therefore, a limit test was selected for the definitive study with a control and a 100 mg/L concentration group. Seven fish (Cyprinus carpio) were used in each test vessel, with one vessel for each of the control and treatment group. The validity criteria for the study was fulfilled as no mortality was observed in the control group, the test conditions remained constant, dissolved oxygen remained above 5 mg/L and the test item concentration was 100% of the nominal value in the treatment group. A reference test with pentachlorophenol was conducted to check the sensitivity of the test system. The 96 hour LC50 was 0.32 mg/L, which is within the historical ranges for the laboratory (0.10 to 0.46 mg/L), conveying that the test system works.

The analytical method of UPLC-RID was used to measure test item concentrations at the start (0 hours) and end of test (96 hours). The concentrations in the control group were not detectable and in the 100 mg/L group, tested concentrations were 100% of nominal. However, there was a lack of information on the analytical method, as no details on the limits of detection, stationary phase or reproducibility were reported. Because the study met all validity criteria specified in the OECD 203 test guideline, and the test concentrations were analysed, the study has been assigned a reliability score of 2.

In conclusion, the 96 hour LC50 was determined as >100 mg/L based on measured concentrations.

Description of key information

A 96-hour LC50 of >100 mg/L was determined for the effects of the test substance on Cyprinus carpio, based on measured concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

Charles River Laboratories (2018) is a GLP-compliant study following OECD guideline 203. A range finding test was conducted with the concentrations of 0.10, 1.0, 10 and 100 mg/L. There was no mortality observed in the control or test concentrations at after 96 hours. Therefore, a limit test was selected for the definitive study with a control and a 100 mg/L concentration group. Seven fish (Cyprinus carpio) were used in each test vessel, with one vessel for each of the control and treatment group. The validity criteria for the study was fulfilled as no mortality was observed in the control group, the test conditions remained constant, dissolved oxygen remained above 5 mg/L and the test item concentration was 100% of the nominal value in the treatment group. A reference test with pentachlorophenol was conducted to check the sensitivity of the test system. The 96 hour LC50 was 0.32 mg/L, which is within the historical ranges for the laboratory (0.10 to 0.46 mg/L), conveying that the test system works.

The analytical method of UPLC-RID was used to measure test item concentrations at the start (0 hours) and end of test (96 hours). The concentrations in the control group were not detectable and in the 100 mg/L group, tested concentrations were 100% of nominal. However, there was a lack of information on the analytical method, as no details on the limits of detection, stationary phase or reproducibility were reported. Because the study met all validity criteria specified in the OECD 203 test guideline, and the test concentrations were analysed, the study has been assigned a reliability score of 2.

In conclusion, the 96 hour LC50 was determined as >100 mg/L based on measured concentrations.