Diallylamine

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 11 January 2016 and 23 February 2016

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and all surviving test groups at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for immediate quantitative 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 water:
The test water used for both the range-finding 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.

Range-finding Test:
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.

In the range-finding test fish were exposed to a series of nominal test concentrations of 1.0, 10 and 100 mg/L. The test item was dissolved directly in test water.

Nominal amounts of test item (20, 200 and 2000 mg) were each separately dissolved in test water and the volume adjusted to 20 litres to give the 1.0, 10 and 100 mg/L test concentrations respectively. Prior to addition of the test organisms, the pH of the 10 and 100 mg/L test preparations was checked and adjusted to 7.6 - 7.7 using hydrochloric acid.

Each prepared concentration was mixed with a flat bladed stirrer for 1 minute to ensure adequate mixing and homogeneity.

In the range-finding test three fish were placed in each test and control vessel and maintained in a temperature controlled room at 13°C to 14°C 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 under static test conditions. Each 20 liter test and control vessel contained 20 liters of test media and was sealed to reduce losses through volitilization. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

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

A sample of each test concentration was taken for chemical analysis at 0 and 24 hours in order to determine the stability of the test item under test conditions.

Definitive Test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

Experimental Preparation:
Nominal amounts of test item (200, 360, 640, 1120 and 2000 mg) were each separately dissolved in test water and the volume adjusted to 20 litres to give the 10, 18, 32, 56 and 100 mg/L test concentration respectively. Prior to addition of the test organisms, the pH of all of the test preparations was checked and adjusted to 7.6 - 7.9 using hydrochloric acid.

Each of the prepared concentrations was mixed with a flat bladed stirrer for 1 minute to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours (fresh media) and 24 and 96 hours (old media).

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 22 January 2016. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 08 February 2016 to 15 February 2016. 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 13°C to 14°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 24 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 5.4 cm (sd = 0.2) and a mean weight of 1.24 g (sd = 0.22) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.43 g bodyweight/liter (static volume).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

water with a total hardness of approximately 140 mg/L as CaCO3

Test temperature:

Temperature was maintained at approximately 14°C throughout the test

pH:

pH was recorded daily throughout the test.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded daily throughout the test.

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56 and 100 mg/L
Mesured: measured test concentrations near nominal

Details on test conditions:

Definitive Test - Exposure Conditions:
As in the range-finding test, 20 liter glass exposure vessels 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 sealed to reduce losses through volitilization and maintained at approximately 14°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 received no auxiliary aeration. 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.

Evaluations:
Test Organism Observations: Any mortalities and sub-lethal effects of exposure were recorded at 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.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Range-finding Test:
The results showed no mortalities at the test concentrations of 1.0 and 10 mg/L. However, mortalities were observed at 100 mg/L.

Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

Chemical analysis of the test preparations at 0 and 24 hours showed that near nominal measured concentrations were obtained, indicating that the test item was stable under test conditions.

Definitive Test:
Verification of Test Concentrations:
Analysis of the test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured test concentrations to be near nominal and so it was considered justifiable to calculate the LC50 values in terms of the nominal test concentrations only. Initial analysis of the 96-Hour samples showed that anomalous results were obtained; however, analysis of the duplicate samples showed that near nominal concentrations were obtained. This was considered to be due to sampling error and was considered not to have had an impact on the outcome of the test.

Mortality Data:
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in 'any other information on results incl. tables' section.

Inspection of the mortality data at 3 and 6 hours and analysis of the mortality data by Probit analysis using Linear Maximum-Likelihood regression at 24, 48, 72 and 96 hours based on the nominal test concentrations gave the following results:

3 h LC50: >100 mg/l
6 h LC50: >100 mg/l
24 h LC 50: 57 mg/L (95% Confidence Limits: Not determined)
48 h LC50: 42 mg/L (95% Confidence Limits: 36 - 49 mg/L)
72 h LC50: 33 mg/L (95% Confidence Limits: Not determined)
96 h LC50: 30 mg/L (95% Confidence Limits: Not determined)

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be 18 mg/L, the lowest test concentration resulting in 100% mortality to be 56 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 32 mg/L. The No Observed Effect Concentration (NOEC) was 18 mg/L.

Sub-Lethal Effects:
Sub-lethal effects of exposure were observed at test concentrations of 56 mg/L and above. These responses were sitting at the bottom of the tank and bubbles stuck to the surface of the fish.

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 in the control and test vessels.

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

Observations on Test Item Solubility:
Throughout the duration of the test, the test preparations were observed to be clear colorless solutions.

Sublethal observations / clinical signs:

Cumulative Mortality Data in the Definitive Test

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0
32	0	0	0	0	3	5	71
56	0	0	3	7*	7	7	100
100	0	0	7	7	7	7	100

*      One fish observed to be dead after approximately 27.5 hours exposure, removed and classed as a mortality for the following observational time point.

Sub-lethal Effects of Exposure in the Definitive Test

Nominal Concentration (mg/L)	Sub-lethal Effects	Time (Hours)
		3	6	24	48	72	96
Control	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
10	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
18	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
32	No abnormalities detected	7/7	7/7	7/7	7/7	4/4	2/2
56	No abnormalities detected	7/7	7/7	2/4	A/D*
	Sat at the bottom of the tank			2/4
100	No abnormalities detected	5/7	5/7	A/D
	Sat at the bottom of the tank	2/7	2/7

*      After approximately 27.5 hours exposure, one of the remaining 4 fish was observed to be dead and the other three observed to have bubbles stuck to their surface. After 48 hours exposure, all fish were observed to be dead.

A/D= 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 nominal test concentrations gave the following results:
96 hours LC50: 30 mg/L
96 hours NOEC: 18 mg/L
96 hours LOEC: 32 mg/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:

Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test item over a range of concentrations of 10, 18, 32, 56 and 100 mg/L for a period of 96 hours at a temperature of approximately14 °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 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results:

Analysis of the test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure ofrainbow trout (Oncorhynchus mykiss)to the test item gave the following results based on the nominal test concentrations:

 

Time Point (Hours)	LC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
96	30	Not determined	18	32

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:

Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test item over a range of concentrations of 10, 18, 32, 56 and 100 mg/L for a period of 96 hours at a temperature of approximately14 °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 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results:

Analysis of the test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure ofrainbow trout (Oncorhynchus mykiss) to the test item gave the following results based on the nominal test concentrations:

 

Time Point (Hours)	LC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
96	30	Not determined	18	32

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

30 mg/L