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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:
08 April 2018 - 10 May 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:
yes
Remarks:
Please see materials and methods section.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not specified
Details on sampling:
Water samples were taken from the control and each test group, from fresh and old media throughout the 96 Hour test period, for quantitative analysis. All samples were stored frozen prior to analysis. 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. Only concentrations of the NOEC (based on mortality) and above were analyzed.
Vehicle:
no
Details on test solutions:
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions

The test water used for both the initial and definitive tests 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.
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 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 16 April 2018 to 23 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 15 C with a dissolved oxygen content of greater than or equal to 9.8 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 4.5 cm (standard deviation = 0.1) and a mean body weight of 0.74 g (standard deviation = 0.12) at the end of the definitive test. Based on the mean body weight value this gave a loading rate of 0.26 g body weight/liter (volume in 24 hours).
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
Nominal and measured concentrations:
Based on the results of the initial test the following test concentrations were assigned to the definitive test: 1.25, 2.5, 5.0, 10 and 20% v/v saturated solution.
Details on test conditions:
In accordance with the recommendations of REACh, the initial 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 or Acute Toxicity to Daphnia magna study 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 (Envigo Study Number MJ04QH) was the lowest of these two EC50 values and hence the initial test was conducted at a single concentration of 15% v/v saturated solution. Due to mortalities in the initial test, a definitive test was conducted at concentrations of 1.25, 2.5, 5.0, 10 and 20% v/v saturated solution.
In this test, an initial loading rate of 100 mg/L was employed, instead of the 50 mg/L that was employed during the media preparation work. This concentration was still considered to have resulted in a saturated solution of the test item, and given that the results correlated with the toxicity profile seen in the definitive test was considered not to have had an impact on the outcome or integrity of the test.

For each 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 approximately 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 test media 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.

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.
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
5.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Initial Test
Cumulative mortality data from the exposure of rainbow trout to the test item during the initial test are given in Table 1 and sub lethal effects of exposure are given in Table 2.
The results showed 100% mortality at the test concentration of 15% v/v saturated solution.
Based on this information test concentrations of 1.25, 2.5, 5.0, 10 and 20% v/v saturated solution were selected for the definitive test.

Definitive Test
Verification of Test Concentrations
Analysis of the freshly prepared 5.0, 10 and 20% v/v saturated solution test preparations at 0 and 72 hours (see Annex 4) showed measured test concentrations to range from 1.5 to 9.0 mg/L. There was no significant change in the measured concentrations at 24 and 96 hours, so the results are based on the fresh measured test concentrations.
Please see table below.

Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 3 and the relationship between percentage mortality and concentration at 96 hours is given in Figure 1.
Inspection of the mortality data at 1, 3, 6, 24 and 48 hours and analysis of the mortality data by the Trimmed Spearman-Karber Procedure at 72 and 96 hours based on the average fresh test concentrations gave the following results:
Please see table below.
The lowest concentration at which 100% mortality occurred was 9.0 mg/L and the highest concentration at which no mortality occurred was 3.7 mg/L.

Sub-Lethal Effects
Sub lethal effects of exposure are given in Table 4.
Sub lethal effects of exposure were observed at test concentrations of 3.7 mg/L and 9.0 mg/L. These responses were sitting on the bottom, increased pigmentation and a loss of equilibrium.
The LOEC was determined to be 3.7 mg/L. The NOEC was 1.9 mg/L.

Nominal Test Concentration
(% v/v Saturated Solution)

Average Fresh Measured Test Concentration (mg/L)

5.0

1.9

10

3.7

20

9.0

Time
(hour)

LC50
(mg/L)

95% Confidence limits
(mg/L)

1

>9.0

-

3

>9.0

-

6

>9.0

-

24

>9.0

-

48

>9.0

-

72

5.8

Not determined due to the nature of the data

96

5.8

Not determined due to the nature of the data

Table3            Cumulative Mortality Data in the DefinitiveTest

Nominal Concentration
(% v/v Saturated Solution)

Average Fresh Measured Concentration

(mg/L)

Cumulative Mortality (Initial Population = 7)
(Hours)

Mortality (%)

1

3

6

24

48

72

96

96
Hours

Control

-

0

0

0

0

0

0

0

0

1.25

-

0

0

0

0

0

0

0

0

2.5

-

0

0

0

0

0

0

0

0

5.0

1.9

0

0

0

0

0

0

0

0

10

3.7

0

0

0

0

0

0

0

0

20

9.0

0

0

0

0

3*

7**

7

100


*Found dead 47 hours after exposure

**Remaining 4 fishfound dead 71 hours after exposure

Table 4-Sub-lethal Effects of Exposure in the Definitive Test

Nominal Concentration
(% v/v Saturated Solution)

Average Fresh Measured Concentration

(mg/L)

Sub-lethal Effects

Time (Hours)

1

3

6

24

47

48

71

72

96

Control

-

No abnormalities detected

7

7

7

7

-

7

-

7

7

1.25

-

No abnormalities detected

7

7

7

7

-

7

-

7

7

2.5

-

No abnormalities detected

7

7

7

7

-

7

-

7

7

5.0

1.9

No abnormalities detected

7

7

7

7

-

7

-

7

7

10

3.7

No abnormalities detected

7

7

7

7

-

7

-

1

1

 

 

Sitting on bottom

0

0

0

0

-

0

-

6

6

 

 

Increased pigmentation

0

0

0

0

-

0

-

6

6

20

9.0

No abnormalities detected

7

7

7

0

0

0

AD

 

 

Sitting on bottom

0

0

0

6

1

0

 

 

Increased pigmentation

0

0

0

7

1

0

 

 

Loss of equilibrium

0

0

0

0

3

4

 


-       =   Not applicable

AD  =  All fish dead

Validity criteria fulfilled:
yes
Conclusions:
Exposure of rainbow trout (Oncorhynchus mykiss) to the test item has been investigated and based on the average fresh measured test concentrations gave the following results:

96hr, LC50-5.8mg/l.
96hr, NOEC-1.9mg/l.
96hr, LOEC-3.7mg/l.
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

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 36 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

In the definitive test, seven fish were exposed to aqueous solutions of the test item, at concentrations of1.25,2.5,5.0,10and20% v/v saturated solution for a period of 96 hours at a temperature of 13 ºC to 14 °C under semi‑static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µmSartorius Sartopore filter, first approximate 2 liters used to pre‑condition the filter was discarded) to produce a saturated solution of the test item with a nominal concentration of100% v/v saturated solution. A series of dilutions was made from this saturated solution to give the required test concentrations of1.25,2.5,5.0,10and20% v/v saturated solution. 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…….

Analysis of the freshly prepared 5.0, 10 and 20% v/v saturated solution test preparations at 0 and 72 hours showed measured test concentrations to range from 1.5 to 9.0 mg/L. There was no significant change in the measured concentrations in the old or expired media at 24 and 96 hours, so the results are based on fresh measured test concentrations.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave the following results based on the fresh measured test concentrations:

Time Point
(Hours)

LC50
(mg/L)

95% Confidence Limits
(mg/L)

0% Mortality

100% Mortality

NOEC
(mg/L)

LOEC
(mg/L)

96

5.8

Not determined due to the nature of the data

3.7

9.0

1.9

3.7


NOEC   =No observedeffect concentration (sub-lethal effects)

LOEC    =Lowest observed effect concentration (sub-lethal effects)

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

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 36 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

In the definitive test, seven fish were exposed to aqueous solutions of the test item, at concentrations of 1.25, 2.5, 5.0, 10 and 20% v/v saturated solution for a period of 96 hours at a temperature of 13 ºC to 14 °C under semi-static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters used to pre-condition the filter was discarded) to produce a saturated solution of the test item with a nominal concentration of 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give the required test concentrations of 1.25, 2.5, 5.0, 10 and 20% v/v saturated solution. 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

Analysis of the freshly prepared 5.0, 10 and 20% v/v saturated solution test preparations at 0 and 72 hours showed measured test concentrations to range from 1.5 to 9.0 mg/L. There was no significant change in the measured concentrations in the old or expired media at 24 and 96 hours, so the results are based on fresh measured test concentrations. Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave the following results based on the fresh measured test concentrations:

Time Point
(Hours)

LC50
(mg/L)

95% Confidence Limits
(mg/L)

0% Mortality

100% Mortality

NOEC
(mg/L)

LOEC
(mg/L)

96

5.8

Not determined due to the nature of the data

3.7

9.0

1.9

3.7


NOEC   =No observedeffect concentration (sub-lethal effects)

LOEC    =Lowest observed effect concentration (sub-lethal effects)

Key value for chemical safety assessment

LC50 for freshwater fish:
5.8 mg/L

Additional information