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Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 09 feb 2017 to 17 may 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
see confidential details
Specific details on test material used for the study:
PHYSICO-CHEMICAL PROPERTIES
- Solubility in water: 2.82 g/L (25 °C)
- pH: 4.3 (1 % (m/v)) Aqueous solution (pKa: -5.0 – 7.8)
- Vapour pressure: 0.0003 hPa (25 °C)
Method: OECD Test Guideline 104
- Specific density: 1.307 g/cm3 (20 °C) Method: OECD Test Guideline 109
Relative density: 1.307 (20 °C)


Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.
Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. The nominal volume per sample was 10 mL. The volume of each sample was recorded.

Sampling schedule for chemical analysis during the test period.
Test Period S Control C1 C2 C3 C4 C5
At the start of exposure R XR XR XR XR XR XR
At the end of exposure R XR XR XR XR XR XR
S Stock solution, kept as a reserve
C1-5 Test solutions
X Sample to be analysed
R Reserve sample

Test solutions

Vehicle:
no
Details on test solutions:
A stock solution of the test item was prepared by dissolving 0.1003 g of the test item in 1000 mL of test medium, resulting in a nominal concentration of 100 mg test item/L. This stock solution was ultrasonicated for 7 min and subsequently stirred for 2 h. The stock solution was clear, without Tyndall effect, and used immediately to prepare the desired test item concentrations. The stock solution was used, undiluted, as the highest test concentration (C5, 100 mg/L). All test solutions were stirred for another 10 min. Visual inspection of the flasks showed clear solutions without Tyndall effect. The following table describes the procedure for preparing the test solutions, and the control for the test.

Preparation of test solutions.
Code of concentration level Nominal test item concentration [mg test item/L] Volume of stock solution used to prepare the test solutions [mL] Code of stock solution used Final volume of the test solutions [mL]
C1 4.27 10.68 C5 250
C2 9.39 23.48 C5 250
C3 20.7 51.7 C5 250
C4 45.5 113.6 C5 250
C5 100 – – 1000

Thereafter the test vessels were filled with the test solutions and adapted to test temperature before the test organisms were added to the test vessels.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
Species: The test system used in this study was Daphnia magna STRAUS (clone M10), a water flea, cultured at ECT Oekotoxikologie GmbH since December 22, 2011.
Origin: The organisms were originally supplied by KU Leuven, Belgium.
Age of parental daphnids: Less than 35 days (25–27 days)
Age of the daphnids at the beginning of the test: Less than 24 hours
Feeding during exposure: None

ACCLIMATION
- Acclimation period: yes
- Acclimation conditions (same as test or not): yes
- Type and amount of food: Algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
- Feeding frequency: 4 times per week
- Health during acclimation (any mortality observed): The health of the Daphnia culture and the overall quality of the test organisms is monitored regularly by using the reference toxicant potassium dichromate (K2Cr2O7) in an acute test design in order to determine the sensitivity of the organisms from the culture and to demonstrate that the test conditions are appropriate.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
none

Test conditions

Hardness:
254 mg/L CaCO3
Test temperature:
19.6-19.9°C
pH:
7.2-7.7
Dissolved oxygen:
8.4-9.6 mg/L
Nominal and measured concentrations:
Nominal: 100, 45.5, 20.7, 9.39 and 4.27 mg test item/L
Geometric mean 0-48h (measured): 95.60, 41.60, 17.30, 8.58, 3.96 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass vessels (nominal volume: 100 mL), covered with watch glasses, were used as test vessels.
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): Twice per week
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM
Test medium: Medium M4 (OECD 2004). Elendt medium M4 was used to dilute the test item and to keep the daphnids during the period of the test. The required volume of medium was prepared within one month prior to use. During storage, the medium was aerated.
- Alkalinity: 0.8 mmol/L HCO3
- Conductivity: 676 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: At start of the test, after temperature adaptation of the test solutions, and at the end of the test the following parameters were measured and recorded in one replicate test vessel of the
control and all treated test concentration: temperature, pH, dissolved oxygen content in % of air saturation value (ASV) and in mg/L.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16/8 h
- Light intensity: Within required ranges (50 – 1000 lx)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality as assessed by immobility of the daphnids was evaluated. Weibull analysis using linear maximum likelihood regression was used to estimate the ECx values.
The biological endpoints were expressed based on the nominal and on mean measured concentrations.
The geometric mean of the measured concentrations of the fresh and aged test solutions was calculated as the squareroot of the product of fresh and aged concentration of a given treatment level.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 0.8; 4; 20 and 100 mg/L
- Results used to determine the conditions for the definitive study: Increased numbers of daphnids (3 to 5 of 5 per replicate) were observed to be less active and/or lying on the vessel bottom at 100 mg/L (24 h) and at 20 mg/L (48 h). Based on the results of a preliminary non-GLP range finding test, the following concentrations in a geometrical series were tested in the definitive test: 4.27, 9.39, 20.7, 45.5 and 100 mg test item/L (spacing factor: 2.2).
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
26.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
12.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Sublethal observations on the mobile daphnids were recorded. An increased number of daphnids lying on the bottom of the test vessels, and daphnids showing reduced swimming activity were observed at 45.5 mg test item/L after 24 hours. After 48 hours of exposure only sporadic sublethal symptoms were observed at 9.39 and 20.7 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid?: yes
The most recent reference test with potassium dichromate (K2Cr2O7) as reference item performed in a separate study (Study No. IDA1611) resulted in an EC50(24 h) of 1.13 mg/L. According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/L and 2.1 mg/L. The toxicity of the reference item is within this range. Therefore the results of this reference test are acceptable and the test conditions are reliable and the sensitivity of the test system could be demonstrated.
Reported statistics and error estimates:
The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

Any other information on results incl. tables

Table 1: Cumulative number of immobilised Daphnia magna after 24 and 48 h.

Nominal concentration

Total number of introduced daphnids

Total number of immobilised daphnids

Immobilised daphnids (% of introduced)

Total number of immobilised daphnids

Immobilised daphnids (% of introduced)

[mg test item/L]

 

[24 h]

[24 h]

[48 h]

[48 h]

Control

20

0

0

0

0

4.27

20

0

0

0

0

9.39

20

2

10

2

10

20.7

20

0

0

0

0

45.5

20

4

20

20

100

100

20

20

100

20

100

Analytical Results

Samples from the test solutions were taken to determine actual levels of the test item in comparison to the nominal concentrations. Analysis of the test item in the samples was performed using an HPLC-MS/MS method. The test item concentrations were stable during the test period between 80 and 120% of nominal concentrations.

Table 2: Summary of measured concentrations during the test.

nominal concentration [mg test item/L]

test period [d]

age of test solution [d]

measured concentration[mg test item/L]

recovery in % of nominal concentration

geometric mean measured concentration over 48 h [mg test item/L]

recovery in % of nominal concentration over 48 h

0 (control)

0

0

< LOD

n.a.

 

 

4.27

0

0

4.50

105

 

 

9.39

0

0

9.66

103

 

 

20.7

0

0

19.8

95.7

 

 

45.5

0

0

47.9

105

 

 

100

0

0

101

101

 

 

0 (control)

2

2

< LOD

n.a.

<LOD

n.a.

4.27

2

2

3.48

81.5

3.96

92.7

9.39

2

2

7.62

81.2

8.58

91.4

20.7

2

2

15.1

72.9

17.3

83.6

45.5

2

2

36.1

79.3

41.6

91.4

100

2

2

90.4

90.4

95.6

95.6

Limit of Quantification (LOQ): 2.0 mg/L; n.d.: Limit of Detection (LOD) 0.6 mg/L; n.a. = not applicable

Analysis of control samples of test medium yielded no residues at or above 30% of the LOQ. Since the geometric mean measured concentrations were between 80 and 120% of nominal, the biological endpoints are expressed based on nominal concentrations. In addition, the biological endpoints are expressed based on the geometric mean measured concentrations.

 

Validity criteria

 All validity criteria were fulfilled as required by the test guideline:

 

Required:

Found:

immobilised daphnids in the control:£10%

0%

dissolved oxygen concentration in control and test vessels
at the end of the test:³
3 mg/L

> = 9.2 mg/L

 

Signs of disease or stress, for example, discoloration or unusual behaviour such as trapping at surface of water, did not exceed 10 per cent of the daphnids in the controls.

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Ethylvanillin is considered as harmful to Daphnia magna (EC50, 48h = 26.2 mg/L (measured concentrations)).
Executive summary:

The 48 hours acute toxicity of ethylvanillin to Daphnia magna was studied under static conditions. Daphnids were exposed to test chemical at nominal concentrations of 0, 4.27, 9.39, 20.7, 45.5 and 100 mg/L (nominal concentrations) or 0, 3.96, 8.58, 17.30, 41.60 and 95.60 mg/L (measured concentrations) for 48 hours. The 48 hours EC50 was 26.2 mg/L (measured concentrations). The 48 hours EC10 based on immobilization was 12.7 mg/L.

Based on the results of this study, ethylvanillin is considered as harmful to Daphnia magna.