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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 12, 2016 - August 22, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Mandelic acid
EC Number:
202-007-6
EC Name:
Mandelic acid
Cas Number:
90-64-2
Molecular formula:
C8H8O3
IUPAC Name:
hydroxy(phenyl)acetic acid
Test material form:
solid
Specific details on test material used for the study:
Designation: Art. 806913
Synonym: DL-Mandelic acid for synthesis
Appearance: White solid
Expiry date: May 31, 20120
Storage: at 20 ± 5 °C in the dark


Preparation of the Test Item:
Before test start and before the test medium renewal the test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 100.0 and 100.2 mg test item into 1000 and 1002 mL test water by intense stirring for 10 minutes. The highest concentration was sterile filtered to minimise bacteria growth during the test and no pH adjustment was conducted. Then, adequate volumes of this solution were diluted with test water to obtain the desired concentrations. The test media were prepared just before introduction of the Daphnia (= start of the test) and test medium renewal on Day 1.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Concentrations:
6.25, 12.5, 25, 50 and 100 mg/L

Sampling method:
Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test and at day 1.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the aged test media of all test concentrations and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of 1.25 with acetonitrile. Additional samples of the control blank and the dilution solvent were taken at test start and test end without any sample treatment.
The vessels for the samples were sterilised before usage.

Sample storage conditions before analysis:
All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Test solutions

Vehicle:
yes
Details on test solutions:
VEHICLE: Reconstituted water "Elendt M4"

Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 8.5
- Total hardness: 250 mg CaCO3/L (calculated)
- Alkalinity: 0.9 mmol/L (calculated)
- The test water was sterile filtered to minimise bacteria growth during the test.

Preparation of the Test Item:
Before test start and before the test medium renewal the test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 100.0 and 100.2 mg test item into 1000 and 1002 mL test water by intense stirring for 10 minutes. The highest concentration was sterile filtered to minimise bacteria growth during the test and no pH adjustment was conducted. Then, adequate volumes of this solution were diluted with test water to obtain the desired concentrations. The test media were prepared just before introduction of the Daphnia (= start of the test) and test medium renewal on Day 1.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna Straus, clone 5
Origin: The Daphnia introduced in the test were taken from ibacon's in-house laboratory culture.
Culture conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny.
Feeding: The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Age: from 2.75 to 21.25 hours old
Acclimation period: same as test

Feeding during test: None

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
250 mg CaCO3/L (calculated)
Test temperature:
20 – 21°C
pH:
6.1 - 8.5
Dissolved oxygen:
6.9 - 9.6 mg/L
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
Measured concentrations: >80% of nominal concentrations
Details on test conditions:
EXPOSURE:
20 Daphnia per control and test concentration, divided into 4 groups of 5 animals, each group in 60 mL test medium

The test was performed as a semi-static test in open vessels.

The duration of exposure was 48 hours. During the exposure period, the mobility of the daphnids was assessed daily, i.e. after 24 and 48 hours.

NO. OF DAPHNIDS:
Control Group: 20 daphnids
6.25 mg/L: 20 daphnids
12.5 mg/L: 20 daphnids
25 mg/L: 20 daphnids
50 mg/L: 20 daphnids
100 mg/L: 20 daphnids

PRE-EXPERIMENT
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
Pre-experiments showed a bacteria growth in the corresponding algae test with this test substance. Preventing bacteria growth and in the pre-test observed degradation of the test item, the test water and the test media were sterile filtered and the test vessels were sterilised.

CONCENTRATION(S)
100, 50, 25, 12.5 and 6.25 mg test item/L and a control

VEHICLE
Reconstituted water (ELENDT M4 medium) was used as vehicle.

Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 6.1 - 8.5
- Total hardness: 250 mg CaCO3/L (calculated)
- O2-Concentration: 6.9 - 9.6 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: 24h (semi-static)


OTHER TEST CONDITIONS
- Photoperiod: 16h light / 8h dark
- Light intensity: light intensity: 560 to 880 lux


Reference substance (positive control):
no
Remarks:
No positive control used in this study. The accuracy and reliability of the test method is demonstrated periodically as recommended by guidelines with potassium dichromate.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: EC50 based on nominal concentrations
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: EC50 based on nominal concentrations
Details on results:
The toxic effect of the test item Art. 806913 to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be ≥ 100 mg test item/L. The 48-hour LOEC was determined to be > 100 mg test item/L and the 48-hour EC50 value was determined to be > 100 mg test item/L.
Results with reference substance (positive control):
In the most recent test with the reference item potassium dichromate performed in January 2016 (study code 88295220) the EC50 after 24 hours was determined to be 1.58 mg test item/L, indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Any other information on results incl. tables

Objective

The purpose of this study was to determine the influence of the test item Art. 806913 on the mobility of Daphnia magna. Young Daphnia (< 24 hours old) were exposed in a semi-static test to the test item for 48 hours, added to test water at a range of concentrations. Under otherwise identical test conditions, different concentrations of the test item result in different percentages of Daphnia being no longer capable of swimming at the end of the test.

Material and Methods

Test Item: Art. 806913; batch no.: S7059413; purity: 99.7 %, according to certificate of analysis

Test Species: Female Daphnia magna, clone 5; 2.75 to 21.25 hours old Source: The Daphnia introduced in the test were taken from ibacon's in-house laboratory culture.

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 20 individuals. The mobility of the Daphnia was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours.

Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentrations: 100, 50, 25, 12.5 and 6.25 mg test item/L and a control

Test Conditions: Water temperature: 20 to 21 °C; pH value: 6.1 to 8.5; dissolved oxygen concentration: 6.9 to 9.6 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 560 to 880 lux; and thus were within the ranges requested by guideline OECD 202

Results

The quantification of the test item Art. 806913 in the test samples was performed using liquid chromatography with UV detection. In the freshly prepared test media at the start of the test and at the renewal of the test media 102% of the nominal test concentrations were found (average of all test concentrations). In the aged test media after 24 and 48 hours test duration, 103% of the nominal value was determined (average of all test concentrations). During the test the test organism were exposed to a mean of 102% of nominal. Therefore, all reported results refer to nominal concentrations.

  Number of daphnids immobilized / exposed    % of daphnids immobilized 
Nominal 24 hours 48 hours 24 hours 48 hours
0 mg/L  0/20  0/20  0%  0% 
6.25 mg/L 0/20  0/20  0%  0% 
12.5 mg/L  0/20  0/20  0%  0% 
25 mg/L  0/20  0/20  0%  0% 
50 mg/L 0/20  0/20  0%  0% 
100 mg/L 0/20  0/20  0%  0% 

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the highest test item concentration of 100 mg test item/L.

Conclusion

The toxic effect of the test item Art. 806913 to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be ≥ 100 mg test item/L. The 48-hour LOEC was determined to be > 100 mg test item/L and the 48-hour EC50 value was determined to be > 100 mg test item/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The toxic effect of the test item Art. 806913 to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be ≥ 100 mg test item/L. The 48-hour LOEC was determined to be > 100 mg test item/L and the 48-hour EC50 value was determined to be > 100 mg test item/L.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 202. The toxic effect of the test item Art. 806913 to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be ≥ 100 mg test item/L. The 48-hour LOEC was determined to be > 100 mg test item/L and the 48-hour EC50 value was determined to be > 100 mg test item/L.

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