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Ecotoxicological information

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:
10 November 2017 to 09 September 2018
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Physical state/Appearance: Brown viscous liquid
Purity: >90%
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and 100 mg/L loading rate WAF test vessels at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis. Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
TEST MEDIUM
Laboratory tap water was dechlorinated by passage through an activated carbon filter and partly softened, 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.

PREPARATION AND APPLICATION OF TEST SOLUTION
Due to the low aqueous solubility and the complex nature of the test item, the test medium was prepared as Water Accommodating Fraction ((WAF) based on a the methods established in a preliminary mixing trial.

A nominal amount of test item was added to the surface of test water to achieve a nominal loading rate of 100 mg/L. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Visual observations of the WAF indicated that a significant amount of dispersed test item was present in the water column and hence, it was considered justifiable to remove the WAF by filtering through a glass wool plug. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first 75 to 100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and no particles or micro-dispersions of test item were observed.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST SYSTEM AND SUPPORTING INFORMATION

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 12 April 2018. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 08 May 2018 to 15 May 2018. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. The water temperature was controlled at approximately 14 °C to 15 °C with a dissolved oxygen content of greater than or equal to 9.7 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 19 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.1 cm (standard deviation = 0.2) and a mean weight of 1.05 g (standard deviation = 0.13) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.37 g body weight/liter.

The test water used for both the range finding and definitive tests was the same as that used to maintain the stock fish.

The diet and test water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
140 mg/L as CaCO3
Test temperature:
14 °C to 15 °C
pH:
7.3 to 8.2
Dissolved oxygen:
9.9 to 10.6 mg O2/L (>60% of air saturated value, ASV)
Nominal and measured concentrations:
Nominal loading rates (WAF) were 0 (control) and 100 mg/L.

Analysis of the freshly prepared test preparations at 0 and 72 hours showed that measured concentrations of 0.10 and 0.080 mg/L as molybdenum (equivalent to 1.2 and 0.94 mg/L as test item) were obtained, respectively; and of the old media at 24 and 96 hours showed that measured concentrations of 0.10 and 0.081 mg/L as molybdenum were obtained, respectively.

The dissolved test item may have been comprised of one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
Details on test conditions:
Based on the results of a range finding test, in which no toxicity was observedt, a "Limit test" was conducted at a single loading rate of 100 mg/L to assess fish toxicity in the definitive test under the same exposure conditions.

Glass exposure vessels (25 - 30 liters) containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

The control group was maintained concurrently under identical conditions, but was not exposed to the test item.

A semi-static test regime was employed in the test involving a daily renewal of the test solutions to aid in maintaining exposure levels and prevent the build-up of nitrogenous waste products.
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
mortality
Details on results:
Exposure of rainbow trout to the test item for 96 hours resulted in an LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary to test at loading rates in excess of 100 mg/L.

There were no sub-lethal effects of exposure observed in seven fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.
Validity criteria fulfilled:
yes
Conclusions:
Exposure of rainbow trout to the test item for 96 hours resulted in an LL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.
It was considered unnecessary to test at loading rates in excess of 100 mg/L.
Executive summary:

A study was performed to the standard OECD Guideline 203 and EU Test Method C.1, under GLP conditions to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). 

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test solutions were prepared as a Water Accommodated Fractions (WAF).

Following a preliminary range finding test, seven fish were exposed to a WAF of the test item at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 14 °C to 15 ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Analysis of the freshly prepared test preparations at 0 and 72 hours showed that measured concentrations of 0.10 and 0.080 mg/L as molybdenum were obtained, respectively, and of the old media at 24 and 96 hours showed that measured concentrations of 0.10 and 0.081 mg/L as molybdenum were obtained, respectively.

The dissolved test item may have been comprised of one or several components of the test item. Given that toxicity cannot be attributed to a single component or a mixture of components, the results were based on nominal loading rates only.

Exposure of rainbow trout to the test item gave LL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

A study was conducted according to OECD Guideline 203 and EC Test Method C.1, under GLP, to assess the acute toxicity of the test item to the freshwater fish, rainbow trout (Oncorhynchus mykiss) using the limit test approach. The 96 hour LL50 value was determined to be greater than 100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) rate was 100 mg/L loading rate WAF (Envigo, 2018).

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information