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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:
Start: 23rd March 2018 Finish 20th April 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)
Version / remarks:
EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification:
1,3-Benzenedimethanamine, reaction products with glycidyl tolyl ether
Batch:
WA 1508
Purity:
not supplied
Physical state/Appearance:
clear colorless viscous liquid
Expiry Date:
01 January 2021
Storage Conditions:
room temperature in the dark
Details on sampling:
Water samples were taken from the control and the 1.1 mg/L loading rate WAF test group, from fresh and old media throughout the 96-Hour test period, for quantitative analysis. Due to a technical oversight the 0-Hour fresh samples were not taken. Duplicate samples at 72 hours (fresh media) and 24 and 96 hours (old media) were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).
In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EL50 value from either the Algal Growth Inhibition study (Envigo study number GR11VD) or Acute Toxicity to Daphnia magna study (Envigo study number PB52WL) is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate. The EL50 value obtained from the Algal Growth Inhibition study was the lowest of these two EL50 values and hence the test was conducted at a single loading rate of 1.1 mg/L.

Experimental Preparation
A nominal amount of test item (24.2 mg) was weighed onto a glass slide and suspended within 22 liters of test water to give the 1.1 mg/L loading rate. 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 95 hours and the mixture allowed to stand for 1-Hour. 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. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.1 mg/L loading rate WAF.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
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 09 March 2018. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 03 April 2018 to 10 April 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 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 20 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 5.3 cm (standard deviation = 0.3) and a mean weight of 1.25 g (standard deviation = 0.21) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.44 g body weight/liter.
The diet and diluent 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:
no
Total exposure duration:
96 h
Test temperature:
14 - 16°C
pH:
7.66
Nominal and measured concentrations:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).
Chemical analysis of the freshly prepared test preparations at 24 and 72 hours and old or expired test preparations at 24, 48 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.053 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.
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:
Experimental Design and Study Conduct
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.
Validation of Mixing Period
Preliminary work (see Annex 2) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.
Definitive Test
In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EL50 value from either the Algal Growth Inhibition study (Envigo study number GR11VD) or Acute Toxicity to Daphnia magna study (Envigo study number PB52WL) is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate. The EL50 value obtained from the Algal Growth Inhibition study was the lowest of these two EL50 values and hence the test was conducted at a single loading rate of 1.1 mg/L.

Experimental Preparation
A nominal amount of test item (24.2 mg) was weighed onto a glass slide and suspended within 22 liters of test water to give the 1.1 mg/L loading rate. 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 95 hours and the mixture allowed to stand for 1-Hour. 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. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.1 mg/L loading rate WAF.

Exposure Conditions
For the control and1.1 mg/L loading rate WAF test concentration, 25 to 30 liter glass exposure vessels containing 20 liters of test media were used. 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 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 under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.

Assessments
Test Organism Observations
Any mortalities and sub-lethal effects of exposure were recorded at 1, 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.
Water Quality Criteria
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of each mixing period.
Chemical Analysis of Test Loading Rates
Water samples were taken from the control and the 1.1 mg/L loading rate WAF test group, from fresh and old media throughout the 96-Hour test period, for quantitative analysis. Due to a technical oversight the 0-Hour fresh samples were not taken. Duplicate samples at 72 hours (fresh media) and 24 and 96 hours (old media) were taken and stored frozen for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in Annex 3.

Data Evaluation
Statistical Analysis
An estimate of the LL50 values was given by inspection of the mortality data.
Data Validation
The results of the test are considered valid if the following criteria are met:
In the control, not more than one of the fish should die or show signs of stress during the 96 hours.
The dissolved oxygen concentration at the end of the test should be ≥60% of Air Saturation Values (ASV) in the control and test vessels.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Details on results:
Validation of Mixing Period
Preliminary investigational work indicated that there was a significant increase in the amount of dissolved test item when the preparation period was extended from 24 to 96 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 95 hours followed by a 1-Hour settlement period.
Definitive Test
Chemical Analysis of Test Loading Rates
Chemical analysis of the test preparations at 24, 48, 72 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.053 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.
Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
Mortality Data
There were no mortalities in seven fish exposed to a 1.1 mg/L loading rate WAF for a period of 96 hours.

The results of the definitive test showed the highest loading rate resulting in 0% mortality to be 1.1 mg/L. The NOEL was considered to be 1.1 mg/L loading rate.

Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in seven fish exposed to a 1.1 mg/L loading rate WAF for a period of 96 hours.
Validation Criteria
The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O2/L) in the control and test vessels.
Water Quality Criteria
Temperature was maintained at 14 °C to 15 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Vortex Depth Measurements
The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.
Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAF.
At the start of the mixing period the 1.1 mg/L loading rate was observed to be a clear colorless water column with test item stuck to glass slide suspended in the water. After 95 hours stirring and a 1-Hour standing period the 1.1 mg/L loading rate was observed to remain as at start of mixing. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the control and 1.1 mg/L loading rate were observed to be clear, colorless
Validity criteria fulfilled:
yes
Remarks:
The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O2/L) in the control and test vessels.
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 LL50 value of greater than 1.1 mg/L loading rate WAF. The NOEL rate was 1.1 mg/L loading rate WAF.
Executive summary:

1.1 Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

1.2 Methods

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

In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EL50 value from either the Algal Growth Inhibition study (Envigo study number GR11VD) or Acute Toxicity to Daphnia magna study (Envigo study number PB52WL) is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate. The EL50 value obtained from the Algal Growth Inhibition study was the lowest of these two EL50 values and hence the test was conducted at a single loading rate of 1.1 mg/L.

Seven fish were exposed to a WAF of the test item, at a single nominal loading rate of 1.1 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.

1.3 Results

Chemical analysis of the freshly prepared test preparations at 24 and 72 hours and old or expired test preparations at 24, 48 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.053 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

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.

Exposure of rainbow trout to the test item gave LL50 values of greater than 1.1 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 1.1 mg/L loading rate WAF.

Description of key information

Key value for chemical safety assessment

LC50 for freshwater fish:
1.1 mg/L

Additional information