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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:
The study was conducted between 17 January 2019 and 25 January 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Batch: Y180510A
Purity: 95.0%
Physical State/Appearance: Black powder
Expiry Date: 31 May 2019
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and 100 mg/L loading rate WAF test vessel, from fresh and old media throughout the 96-Hour test period. Water samples were taken from the control test vessel at 0 hours from unstirred media, at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for immediate quantitative analysis. Additional single samples were also taken at 24 and 48 hours (fresh), 48 and 72 hours (old) and frozen for analysis if required.

Duplicate sets of samples at 0 and 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:
A nominal amount of test item (2200 mg) was added to the surface of 22 liters of test water to give the 100 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. Observations made on 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 dispersed material by filtering the WAF through a glass wool plug (2 to 4 cm in length). 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 filtered was discarded) to give the 100 mg/L loading rate WAF. Observations after filtering through a glass wool plug showed cloudy dispersions or black micro-dispersions of test item, therefore, the WAF was filtered through filter paper. Following filtration through filter paper, no micro-dispersions were observed.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 24, 72 and 96 hours.
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 14 November 2018. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 14 January 2019 to 21 January 2019. 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 with a dissolved oxygen content of greater than or equal to 9.5 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 27 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 = 0.3) and a mean weight of
1.08 g (standard deviation = 0.21) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.38 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:
yes
Total exposure duration:
96 h
Hardness:
140 mg/L as CaCO3
Test temperature:
13 °C to 14 °C
pH:
pH 7.4 - 8.2
Dissolved oxygen:
9.9 - 10.5 mg O2/L
Nominal and measured concentrations:
100 mg/L loading rate WAF

Chemical analysis of the 100 mg/L loading rate WAF at 0, 24, 72 and 96 hours showed measured silicon concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 1.0 mg/L as silicon. This does not infer that no silicon was in solution, just that any dissolved test item was at a concentration of less than the LOQ. A sample of the test media before stirring was taken to compare the silicon levels with the stirred media to see if there was an increase from the stirring process (as was found in the Acute Toxicity to Daphnia magna study - Envigo study number LB68DJ). The measured silicon concentration in the unstirred media sample was also less than the LOQ.
Due to background levels of silicon present in dechlorinated tap water the LOQ for the analytical method for this study was higher (at 1.0 mg/L as Si) than that established for the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study (Envigo study numbers NQ61QY and LB68DJ respectively).

The dissolved test item may have been one or several components of the test item. 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.
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
The test item contained more than one component, so verification of the test concentration was based on analysis of the marker component silicon as an indicator of the test item presence. Preliminary work (see Annex 3) was carried out to determine whether stirring for a prolonged period produced significantly higher measured silicon 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 or Acute Toxicity to Daphnia magna study 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. Therefore, as the EL50 value obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study (Envigo study numbers NQ61QY and LB68DJ respectively) were greater than 100 mg/L loading rate WAF, the test was conducted at a single loading rate of 100 mg/L loading rate WAF to
ensure that toxicity was not observed at this loading rate.

Exposure Conditions
For the control and 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, containing the test preparations. The test vessels were then covered to reduce evaporation and maintained at 13 °C to 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness, and 20 minute dawn and dusk transition periods, for a period of 96 hours. The contents of the test vessels were aerated. 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.

Data Evaluation
An estimate of the LL50 values was given by inspection of the mortality data.

Validation Criteria
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.:
> 100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality and sub-lethal effects
Details on results:
Mortality Data
There were no mortalities in seven fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours. The results of the definitive test showed the 100 mg/L loading rate resulted in 0% mortality giving a NOEL of 100 mg/L loading rate WAF. The LL50 was greater than 100 mg/L.

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

Sub-Lethal Effects
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.

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 (≥9.9 mg O2/L) in the control and test vessels.

Water Quality Criteria
Temperature was maintained at 13 °C to 14 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Vortex Depth Measurements
The vortex depth recorded at the start and end of each mixing period, 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 100 mg/L preparation was observed to be a clear colorless water column with test item at the surface and slowly dispersing through water column. After 95 hours stirring and a 1-Hour standing period the 100 mg/L preparation was observed to be a grey cloudy dispersion with test item at sides of vessel and at the surface.
Examination of the WAF showed undissolved test item present and therefore it was considered justifiable to remove the dispersed material by filtering the WAF through a glass wool plug (2 to 4 cm in length). Observations after filtering through glass wool plug showed a cloudy dispersion or black micro-dispersions; therefore the WAF was filtered through filter paper. Following filtration through filter paper, no micro-dispersions were observed.
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
Sublethal observations / clinical signs:

Cumulative Mortality Data in the DefinitiveTest

 

 

Nominal Loading Rate (mg/L)

 

Cumulative Mortality (Initial Population = 7)

Mortality (%)

1

Hour

3 Hours

6

Hours

24

Hours

48

Hours

72

Hours

96

Hours

96

Hours

Control

0

0

0

0

0

0

0

0

100

0

0

0

0

0

0

0

0

Sub-lethal Effects of Exposure in the DefinitiveTest

 

Nominal Loading Rate (mg/L)

 

 

Sub-lethal Effects

Time(Hours)

1

3

6

24

48

72

96

Control

No abnormalities detected

7

7

7

7

7

7

7

100

No abnormalities detected

7

7

7

7

7

7

7

Water Quality Measurements

 

 

Nominal Loading Rate (mg/L)

Time (Hours)

0 Hours (Fresh Media)

24 Hours (Old Media)

pH

mg O2/L

Temperature

ºC

pH

mg O2/L

Temperature

ºC

Control

7.4

10.3

14

7.9

10.0

13

100

7.5

10.3

14

7.9

9.9

13

 

 

Nominal Loading Rate (mg/L)

Time (Hours)

24 Hours (Fresh Media)

48 Hours (Old Media)

pH

mg O2/L

Temperature

ºC

pH

mg O2/L

Temperature

ºC

Control

7.6

10.4

14

8.2

10.1

13

100

7.7

10.4

14

8.2

9.9

13

 

 

Nominal Loading Rate (mg/L)

Time (Hours)

48 Hours (Fresh Media)

72 Hours (Old Media)

pH

mg O2/L

Temperature

ºC

pH

mg O2/L

Temperature

ºC

Control

8.0

10.3

14

7.9

10.2

13

100

7.9

10.4

14

7.9

10.1

13

 

 

Nominal Loading Rate (mg/L)

Time (Hours)

72 Hours (Fresh Media)

96 Hours (Old Media)

pH

mg O2/L

Temperature

ºC

pH

mg O2/L

Temperature

ºC

Control

7.8

10.6

13

7.8

10.1

13

100

7.7

10.5

13

7.8

10.0

13

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 LL50 value of greater than 100 mg/L loading rate WAF. The NOEL was 100 mg/L loading rate WAF.
Executive summary:

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.

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 or Acute Toxicity to Daphnia magna study 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.  Therefore, as the EL50 value obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study (Envigo study numbers NQ61QY and LB68DJ respectively) were greater than 100 mg/L loading rate WAF, the test was conducted at a single loading rate of 100 mg/L loading rate WAF to ensure that toxicity was not observed at this loading rate.  

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 13 °C to 14 °C 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.

Results

Chemical analysis of the test preparations was conducted using inductively coupled plasma with mass spectrometric detection (ICP-MS) for the presence of silicon in solution. Analysis of the 100 mg/L loading rate WAF preparation at 0, 24, 72 and 96 hours showed measured silicon concentrations of less than the limit of quantification (LOQ) of the analytical method which was determined to be 1.0 mg/L as silicon.  This does not infer that no silicon was in solution, just that any dissolved silicon was at a concentration of below the quantifiable level.

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.

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 and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

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.

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 or Acute Toxicity to Daphnia magna study 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.  Therefore, as the EL50 value obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study (Envigo study numbers NQ61QY and LB68DJ respectively) were greater than 100 mg/L loading rate WAF, the test was conducted at a single loading rate of 100 mg/L loading rate WAF to ensure that toxicity was not observed at this loading rate.  

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 13 °C to 14 °C 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.

Results

Chemical analysis of the test preparations was conducted using inductively coupled plasma with mass spectrometric detection (ICP-MS) for the presence of silicon in solution. Analysis of the 100 mg/L loading rate WAF preparation at 0, 24, 72 and 96 hours showed measured silicon concentrations of less than the limit of quantification (LOQ) of the analytical method which was determined to be 1.0 mg/L as silicon.  This does not infer that no silicon was in solution, just that any dissolved silicon was at a concentration of below the quantifiable level.

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.

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 and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

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