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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
The test medium samples were analyzed analytically immediately after sampling.
Vehicle:
no
Details on test solutions:
TEST MEDIUM
All testing was performed using a synthetic medium. Chemical reagents used for the preparation of this dilution water were of “analytical grade”; it is prepared according to the protocol as described below to get the concentration specified in the test guideline:

The following solutions were prepared:
- CaCl2, 2H2O: 409 g per 1L of deionised water,
- MgSO4, 7H2O: 171.3 g per 1L of deionised water,
- NaHCO3: 90.8 g per 2 L of deionised water,
- KCl: 7.9 g of per 0.5L of deionised water,

All these solutions were then mixed with around 1390L of deionised water to get the following final recommended concentrations:
- CaCl2, 2H2O: 294 mg/L
- MgSO4, 7H2O: 123 mg/L
- NaHCO3: 64.7 mg/L
- KCl: 5.7 mg/L

TEST SOLUTION PREPARATION (RANGE-FINDING TEST)
Tests solutions were prepared from dilutions of a stock solution: a known volume of test item (72.7 µL) was poured into a volumetric flask, the volume was then made up to 100 mL with test medium. The solution was kept under high speed stirring at room temperature during approximately 15 minutes with a magnetic stir bar. Due to the presence of undissolved particles, this solution was filtered through 0.45 µm filter before dilution. In accordance with the Sponsor's representative, the pH of the test item stock solution was measured at pH 10.4 and adjusted to pH 8.3 before dilutions. Daphnids were exposed under static conditions (i.e. without test solutions renewal) at the following nominal test concentrations: 1.0, 10.0, 50.0 and 100.0 mg/L. Two vessels were prepared at each test concentration and the control group; each vessel contained 5 daphnids.

TEST SOLUTION PREPARATION (DEFINITIVE TEST)
Based on the results of the range-finding test, the definitive test was conducted under static conditions at the following nominal test concentrations: 25.0, 50.0, 100.0, 200.0, 400.0 and 800.0 mg/L. The range of concentrations was obtained from dilutions of a stock solution prepared as follows: a known volume of test item (1163 µL) was poured into a volumetric flask, the volume was then made up to 1000 mL with test medium. The solution was kept under high speed stirring at room temperature during approximately 15-20 minutes with a magnetic stir bar. Due to the presence of undissolved particles, this solution was filtered through 0.45 µm filter before dilution. The pH of the test item stock solution was adjusted to pH 7.9 before dilutions. Four vessels were prepared at each test concentration and the control group; each vessel contained 5 daphnids.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test organisms used for this study was Daphnia magna Straus (Cladocera, Crustacea), clone 5, bred within the laboratory by acyclical parthenogenesis in a synthetic medium. D. magna cultures were fed with a mixture of the microalgae Chlorella vulgaris and Pseudokirchneriella subcapitata. The neonates used were less than 24h old at test initiation (selected by filtration) and were not first brood progeny. Test animals were not fed during the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
240 mg/L as CaCO3
Test temperature:
20.3°C - 20.5 °C (min-max)
pH:
7.1 - 8.0 (min - max)
Dissolved oxygen:
8.2 - 8.9 mg/L (min - max)
Nominal and measured concentrations:
Nominal: control ; 25 ; 50 ; 100 ; 200 ; 400 ; 800 mg/L.
Measured: 0 ; 35.8 ; 67.8 ; 107.0 ; 220.4 ; 459.3 ; 938.1 mg/L
Details on test conditions:
ENVIRONMENTAL CONDITIONS
Culture and test vessels containing D. magna (20 daphnids per tested concentration for the definitive test) were incubated with a photo period of 8 hours of darkness per 24h at a temperature ranging from 18°C to 22°C but remaining constant within ± 1°C during the test. Test vessels were made of clear glass tubes (around 20 mL capacity) and covered to minimise evaporation and avoid the entry of dust into the solutions. At least 3 mL of exposure solution was provided for each neonate. The control treatment was maintained under the same conditions as the treated groups, except it did not contain any test item. No auxiliary substance was used.

OBSERVATIONS
D. magna were observed after 24 hours and 48 hours (end of the exposure period). Organisms which were unable to swim within 15 sec following gentle agitation of the test container were considered to be immobile. The number of immobile Daphnia was recorded in each group at 24 hours and 48 hours after the start of the experiment. In addition, any abnormal behaviour was reported. For the definitive test, pH and dissolved oxygen concentrations were measured in a single vessel for each tested concentration at the test initiation and thereafter at the end of the 48h exposure. The total hardness was determined at the start of the test in the control group. The temperature was continuously recorded throughout the test, in a separated flask maintained within the same conditions.
Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
309 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
DEFINITIVE TEST
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, no precipitation nor visible undissolved particles was observed at the end of the test. The light intensity was measured in a range from 620 to 800 lux.
The numbers of immobile D. magna recorded over the test period are presented in Table 1. Test item concentration results are presented in Table 2. Chemical analysis of test samples taken indicated that measured concentrations of the test item did not remained within 80-120% of the nominal concentrations: recovery rates are superior to 100% meaning that measured concentrations are higher than nominal concentrations.
Chemical analysis of test samples taken at 48h indicated that measured concentrations of the test item remained within 80-120% of the initial measured concentrations, the stability of the substance was thus confirmed. Based on these results, the exposure concentrations were based on the initial measured concentrations. 48h-EC50 was 309 mg/L.
Results with reference substance (positive control):
The sensitivity of the test system and the methodology are evaluated monthly by performing an acute Daphnia magna toxicity test on potassium dichromate. The most recent values of 24h-EC50, obtained on September 2017 was as follows: 24h-EC50 = 0.94 mg/L. The ISO 6341 norm has a validity criterion specifying that the reference item EC50 must be in the 0.6 to 2.1 mg/L range.
Reported statistics and error estimates:
Raw data were treated according to the recommendations of the guideline using Software ToxRatPro Version 3.2.1. The measured concentration of the substance being tested were not satisfactorily maintained within ± 20% of the nominal concentrations. However, the test item concentrations measured at the end of the test period (i.e. 48h) were satisfactorily maintained within ± 20% of the initial measured concentrations. Analysis of the results was thus based on the initial measured concentrations.

Table 1: Cumulative immobilisation (Definitive test)

Test solution

(mg/L)

Total daphnids

at T0

Immobile daphnids

Mobility inhibition at 48h(%)

24h

48h

Control

20

0,0

0,0

0%

25.0

20

0,0

0,0

0%

50.0

20

0,0

0,0

0%

100.0

20

0,0

0,0

0%

200.0

20

0,0

0,0

0%

400.0

20

15,0

20,0

100%

800.0

20

20,0

20,0

100%

Table 2: Test item determination at T0 (Definitive test)

Test solution

(mg/L)

Test item concentration(mg/L)

Mean

SD

RSD (%)

Recovery rate (%)

Measure 1

Measure 2

Measure 3

Control

< LD

< LD

< LD

NA

NA

NA

NA

25.0

36.4

35.5

35.7

35.8

0.5

1.3

143.4

50.0

70.1

66.2

67.1

67.8

2.1

3.0

135.6

100.0

109.5

106.5

105.0

107.0

2.3

2.1

107.0

200.0

221.4

222.6

217.1

220.4

2.9

1.3

110.2

400.0

463.5

455.0

459.5

459.3

4.3

0.9

114.8

800.0

1017.0

911.6

885.7

938.1

69.6

7.4

117.3

< DL (3.22 mg/L): concentration lower than the Detection Limit

< QL (10.8 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

 

Table 3: Test item determination at 48h (Definitive test)

Test solution

(mg/L)

Test item concentration(mg/L)

Mean

SD

RSD (%)

Recovery rate (%)

Measure 1

Measure 2

Measure 3

Control

< LD

< LD

< LD

NA

NA

NA

NA

25.0

26.2

37.0

33.2

32.2

5.5

17.0

128.6

50.0

62.9

62.6

56.6

60.7

3.6

5.9

121.4

100.0

109.4

ND

ND

109.4

NA

NA

109.4

200.0

221.0

ND

ND

221.0

NA

NA

110.5

400.0

473.4

495.8

540.9

503.4

34.4

6.8

125.8

800.0

986.2

ND

1026.2

1006.2

28.3

2.8

125.8

< DL (3.22 mg/L): concentration lower than the Detection Limit

< QL (10.8 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

ND: Not Determined due to sample preparation failure

Table 4: Test item determination – Concentrations used for statistical analysis (Definitive test)

Test solution

(mg/L)

Test item concentration(mg/L)

Deviation of 48h measured

conc. from 0h (%)

Used conc.

(mg/L)*

T0

T48h

 Control

< LD

< LD

NA

NA

25.0

35.8

32.2

-10.3

35.8

50.0

67.8

60.7

-10.4

67.8

100.0

107.0

109.4

2.2

107.0

200.0

220.4

221.0

0.3

220.4

400.0

459.3

503.4

9.6

459.3

800.0

938.1

1006.2

7.3

938.1

< DL (3.22 mg/L): concentration lower than the Detection Limit

< QL (10.8 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

*Concentrations extrapolated as initial measured concentrations

 

Validity criteria fulfilled:
yes
Conclusions:
ethyl(methyl)amine 48-EC50 is 309 mg/L for daphnid immobilization.
Executive summary:

This study was designed to determine the effects of the test item to Daphnia magna over a 48h period with static conditions, according to the OECD 202 Guideline. The tests solutions were prepared from dilutions of a stock solution. 

The measured concentration of the substance being tested were not satisfactorily maintained within ± 20% of the nominal concentrations. However, the test item concentrations measured at the end of the test period (i.e. 48h) were satisfactorily maintained within ± 20% of the initial measured concentrations. Therefore, initial measured concentrations were used to determine 48h-EC50 = 309 mg/L. The test met the validity criteria of the test guideline.

Description of key information

ethyl(methyl)amine 48 h-EC50 is 309 mg/L for daphnid immobilization.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
309 mg/L

Additional information