Diallylamine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 18 August 2015 and 05 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours .

Vehicle:

no

Details on test solutions:

Range- finding Tests
In the initial range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give the further test concentrations of 0.10, 1.0 and 10 mg/L. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. During the initial range finding test, pH values in excess of the range of 6-9 given in the guideline were measured. Consequently, a second range finding test was conducted with the adjustment of pH.

In the second range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give the required test concentrations of 0.10, 1.0 and 10 mg/L. Prior to addition of the daphnia, the pH of the test preparations was measured and if necessary, adjusted to 8.0 using 1.0 M hydrochloric acid. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L. Given that no significant differences were observed between the standard or pH adjusted tests, it was considered unnecessary to adjust the pH of the test preparations during the definitive test.

Experimental Preparation
A nominal amount of test item (200 mg) was dissolved in test water and the volume adjusted to
2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give the further test concentrations of 1.0, 3.2, 10 and 32 mg/L.
Each prepared concentration was inverted several times 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 48 hours.

Exposure Conditions
In the definitive test 100 mL stoppered conical flasks containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then sealed to prevent losses through volatilization and maintained in a temperature controlled room at 21 ºC to 22 ºC with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

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

The test preparations were not renewed during the exposure period.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures. Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 ºC. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Reconstituted Water – Elendt M7 Medium
Ingredient Final Concentration
(mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075

The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

No data

Test temperature:

Water temperature was recorded daily throughout the test. The temperature was maintained at 21ºC-22ºC throughout the test. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

The pH was recorded at the start and termination of the test. The pH was measured using a Hach Flexi handheld meter.

Dissolved oxygen:

Dissolved oxygen concentrations was recorded at the start and termination of the test. Dissolved oxygen concentration was measured using a Hach Flexi handheld meter.

Salinity:

Not applicable as freshwater used

Nominal and measured concentrations:

Following preliminary range-finding tests, the concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were used. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

Details on test conditions:

Exposure Conditions
In the definitive test 100 mL stoppered conical flasks containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then sealed to prevent losses through volatilization and maintained in a temperature controlled room at 21 C to 22 C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item. The test preparations were not renewed during the exposure period.

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the four pooled replicates at 48 hours for quantitative analysis. Samples were analyzed immediately. Duplicate samples were taken and stored frozen for further analysis if necessary.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilization

Remarks on result:

other: 95% CL: Not determined

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilization

Remarks on result:

other: 95% CL: 22-40 mg/L

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilization

Remarks on result:

other: 95% CL: Not determined

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilization

Remarks on result:

other: 95% CL: 22-40 mg/L

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilization

Remarks on result:

other: 95% CL: Not determined

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilization

Remarks on result:

other: 95% CL: 22-40 mg/L

Details on results:

Range-finding Tests
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding tests are given in Table 1 and Table 2. No significant immobilization was observed in either range finding test at the test concentrations of 0.10, 1.0 and 10 mg/L, however, immobilization was observed at 100 mg/L. In the initial range finding test, 1 daphnia was observed to be immobilized and 3 daphnia were observed to be trapped at the surface of the media in the 10 mg/L test concentration.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test. Given that results both with and without pH adjustment were not significantly different, the definitive test was conducted without the adjustment of the pH in order to give a better representation of the intrinsic properties of the test item.

Chemical analysis of the test preparations in the initial range finding test at 0 and 48 hours (see Appendix 5) showed measured test concentrations to be near nominal indicating that the test item was stable under test conditions.

Definitive Test
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.
Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 3.
One immobilized daphnia was observed in the control group, however, as no more than 10% immobilization occurred, this was considered to be due to natural causes and as such considered to have no impact on the outcome of the test.

Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/L) 95% Confidence limits
(mg/L)

24 42 Not determined
48 30 22 - 40


The No Observed Effect Concentrations after 24 and 48 hours exposure were 32 and 10 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 100 and 32 mg/L respectively. No sub-lethal effects of exposure were observed throughout the test.

The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
Temperature was maintained at 21 ºC to 22 ºC throughout the test. While there were no treatment related differences for oxygen concentration, concentration dependent differences in pH were observed throughout the test.
Throughout the test the light intensity was observed to be in the range of 571 to 584 Lux.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control (Study Number 41500252) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:


Time Point EC50 95% Confidence Limits No Observed Effect Concentration Lowest Observed Effect Concentration
(Hours) (mg/L) (mg/L) (NOEC) (mg/L) (LOEC) (mg/L)

24 1.1 Not possible to determine 0.56 1.0

48 0.75 0.69-0.82 0.56 1.0



The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Table1     Cumulative Immobilization Data and Observations in the Initial Range-finding Test

Nominal Concentration (mg/L)	Observations (Initial Population: 10 Per Replicate)
	24 Hours		48 Hours
	Cumulative Immobilized Daphnia	Observations	Cumulative Immobilized Daphnia	Observations
Control	0	10 N	0	10 N
0.10	0	10 N	0	10 N
1.0	0	10 N	0	10 N
10	0	10 N	1	3 T 6 N
100	10	A/I	10	A/I

N = No sub-lethal effects observed

T = Trapped at surface

A/I = All daphnia immobilized

Table2     Cumulative Immobilization Data and Observations in the Second Range-finding Test

Nominal Concentration (mg/L)	Observations (Initial Population: 10 Per Replicate)
	24 Hours		48 Hours
	Cumulative Immobilized Daphnia	Observations	Cumulative Immobilized Daphnia	Observations
Control	0	10 N	0	10 N
0.10	0	10 N	0	10 N
1.0	0	10 N	0	10 N
10	0	10 N	0	10 N
100	10	A/I	10	A/I

N = No sub-lethal effects observed

A/I = All daphnia immobilized

Table3     Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.0	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	0	0	0	1	1	5	5 N	5 N	5 N	4 N
100	5	5	5	5	20	100	A/I	A/I	A/I	A/I

Nominal Concentration (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	1	1	5	5 N	5 N	5 N	4 N
1.0	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	1	0	1	5	5 N	5 N	4 N	5 N
32	1	3	3	3	10	50	4 N	2 N	2 N	2 N
100	5	5	5	5	20	100	A/I	A/I	A/I	A/I

R₁– R₄= Replicates 1 to 4

N = No sub-lethal effects observed

A/I = All daphnia immobilized

Validity criteria fulfilled:

yes

Conclusions:

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:

Time Point EC50 95% Confidence Limits No Observed Effect Concentration Lowest Observed Effect Concentration
(Hours) (mg/L) (mg/L) (NOEC) (mg/L) (LOEC) (mg/L)

48 30 22-40 10 32

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

Methods…….

Following preliminary range-finding tests, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of 21°C to 22°C under static test conditions. The number of immobilized daphnia and any adverse reactions were recorded after 24 and 48 hours.

 

Results…….

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

 

Exposure of Daphnia magna to the test item gave the following results:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	30	22 - 40			10	32

 

 

 

Description of key information

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods…….

Following preliminary range-finding tests, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of 21°C to 22°C under static test conditions. The number of immobilized daphnia and any adverse reactions were recorded after 24 and 48 hours.

Results…….

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations.

 

Exposure of Daphnia magna to the test item gave the following results:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	30	22	-	40	10	32

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

30 mg/L