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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:
4 February 2018 - 20 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Appearance: Viscous amber liquid (determined by Charles River Den Bosch)
- Purity/Composition: >85%
- Test item storage: At room temperature

Additional information
- Test Facility test item number: 209080/A
- Purity/Composition correction factor: No correction factor required
- Chemical name (IUPAC), synonym or trade name: Glyceryl Triacetyl Hydroxystearate
- CAS number: 139-43-5
- Molecular formula: C63H116O12
- Molecular weight: 1065.6
- Highly reactive to water: Not indicated
- Highly reactive to oxygen: Not indicated
- Solubility in water: Insoluble
- Stability in water: Stable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
- Frequency: at t=0 h and t=48 h
- Volume: 1.0 mL from the approximate centre of the test vessels.
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 1.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of Hetester HCA tested was a viscous amber liquid with a purity >85% which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were transferred to separation funnels and subsequently allowed to settle for a period of one hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were tapped from the separation funnels and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
Any residual volumes were discarded.
The water solubility of Hetester HCA at 20°C was determined to be <1.5 µg/L, using the slow-stirring flask method (Test Facility Study No. 20136823).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Test System
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- Reason for selection: This system has been selected as an internationally accepted invertebrate species.
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Breeding
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
- Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Composition of medium M7
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):

Macro salts:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements:
B 0.125 mg/L
Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rb and Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025 mg/L
Se 0.0010 mg/L
V 0.0003 mg/L
Na2EDTA.2H2O 2.5 mg/L

Macro nutrients:
Na2SiO3.9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L

Vitamins:
Thiamine 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L
The hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
CaCO3: 180 mg/l
Test temperature:
19 - 20 °C
pH:
8.0 - 8.1
Dissolved oxygen:
8.3 - 9.4
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
100 mg/l loading rate
Details on test conditions:
Test Concentrations
- Test solution: WAFs individually prepared at loading rates of 1.0, 10 and 100 mg/L
- Control: Test medium without test item or other additives.

Test Procedure and Conditions
- Test duration: 48 hours
- Test type: Static
- Test vessels: 60 mL, all-glass
- Medium: Adjusted ISO medium
- Number of daphnids: Control and limit concentration: 20 per test group. WAFs prepared at 1.0 and 10 mg/L: 10 per test group
- Loading: 5 per vessel containing 50 mL of test solution
- Light: 16 hours photoperiod daily
- Feeding: No feeding
- Aeration: No aeration of the test solutions.
- Introduction of daphnids: 12 minutes after preparation of the test solutions.

Sampling for Analysis of Test Concentrations
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
- Frequency: at t=0 h and t=48 h
- Volume: 1.0 mL from the approximate centre of the test vessels.
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 1.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Measurements and Recordings
- Immobility (including mortality): At 24 hours and at 48 hours.
- pH and dissolved oxygen: At the beginning and at the end of the test, for the control and the WAF prepared at 100 mg/L
- Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Positive reference substance
The objective of the study was to evaluate potassium dichromate (K2Cr2O7) for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure (Charles River Den Bosch Study Number 20133130).
The study procedures described in this report were based on the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004 and the ISO International Standard 6341.
Start of first exposure: 02 Jan 2018
Completion last exposure: 04 Jan 2018
The reference test was carried out to check the sensitivity of the test system as used by Charles River Den Bosch. Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a control. Twenty daphnids were exposed per concentration.
The reference item, potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K44879664) was obtained from Merck, Darmstadt, Germany.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Limit of water solubility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Limit of water solubility
Details on results:
MEASURED CONCENTRATIONS
Samples taken from the WAF prepared at a loading rate of 100 mg/L were analysed. The measured concentration at the start of the test was 3.5 mg/L which decreased to 1.9% of initial at the end of the test. Based on these results, effects parameters were based on the average exposure concentration of 0.48 mg/L calculated at the limit concentration (see Table 1).
The measured concentration at the start and at the end of the test exceeded the water solubility limit, determined to be <1.5 µg/L (Test Facility Study No. 20136823). It can nevertheless be stated that testing was ultimately performed at the maximum soluble concentration of test item in test medium with a TWA concentration of 0.48 mg/L.

IMMOBILITY
Table 2 shows the responses recorded during the test. No immobility or other adverse effects were observed at any of the test concentrations throughout the test. After 48 hours of exposure, a floating layer was observed at the WAF prepared at a loading rate of 100 mg/L, indicating probable over-saturation at the limit concentration.
The responses recorded in this test allowed for reliable determination of an EC50.

DETERMINATION OF EFFECT CONCENTRATIONS
Table 3 shows the effect parameters based on the average exposure concentration of 0.48 mg/L calculated at the limit concentration.

EXPERIMENTAL CONDITIONS
The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 4. These test conditions remained within the limits prescribed by the study plan (pH: 6 9, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the test).
The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).
Reported statistics and error estimates:
Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate:
Concentration
K2Cr2O7 Number
Exposed % immobile
24h 48h Expected response (%)
After 48 hours 1
(mg/L) Minimal Maximal
control 20 0 0 0 102
0.10 20 0 0 0 10
0.18 20 0 0 0 10
0.32 213 0 5 0 30
0.56 20 0 55 0 100
1.0 20 80 100 40 100
1.8 20 100 100 100 100
1 Based on historical data of the previous years (n>60).
2 A maximum response of 10% does not invalidate the results of the test.
3 Unintentionally, an extra daphnid was added to one of the test vessels.
In conclusion, the actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
The 24h-EC50 was 0.87 mg/L with a 95% confidence interval between 0.80 and 0.95 mg/L.
The 48h-EC50 was 0.54 mg/L with a 95% confidence interval between 0.49 and 0.61 mg/L.

Any other information on results incl. tables

Table 1: Average Exposure Concentration Versus Loading Rate

Hetester HCA

Loading rate (mg/L)

Measured concentrations (mg/L)

Average exposure conc. (mg/L)

t=0h

t=48h

100

3.529

0.0660

0.48

Table 2: Number of Introduced Daphnids and Incidence of Immobility

Time (h)

Replicate

Hetester HCA; Loading rate (mg/L)

Control

1.0

10

100 (0.48)

0

A

5

5

5

5

B

5

5

5

5

C

5

5

D

5

5

Total introduced

20

10

10

20

24

A

0

0

0

0

B

0

0

0

0

C

0

 

 

0

D

0

 

 

0

Total immobilised

0

0

0

0

Effect %

0

0

0

0

 

 

 

 

48

A

0

0

0

0

B

0

0

0

0

C

0

 

 

0

D

0

 

 

0

Total immobilised

0

0

0

0

Effect %

0

0

0

0

( ) – average exposure concentration (mg/L).

Table 3: Effect Parameters

Parameter Hetester HCA
Average exposure conc. (mg/L)
24h,48h-EC50 >0.48


Table 4: pH and Oxygen Concentrations (mg/L) During the Test

Hetester HCA Start (t=0 h) End (t=48 h)
Loading rate (mg/L) pH O2 pH O2
Control 8 9.4 8.1 8.5
100 (0.48) 8.1 8.7 8 8.3

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Executive summary:

The objective of the study was to evaluate Hetester HCA for its ability to generate acute toxic effects on the mobility ofDaphnia magnaduring an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004.In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of EC 205-363-0 tested was a viscous amber liquid with a purity of >85% and not completely soluble in test medium at the loading rates initially prepared.

A combined limit/range-finding test was performed. Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0, 10 and 100 mg/L and used as test concentrations.

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from the WAF prepared at a loading rate of 100 mg/L were analysed.The measured concentration at the start of the test was 3.5 mg/L which decreased to 1.9% of initial at the end of the test. Based on these results, effects parameters were based on the average exposure concentration of 0.48 mg/L calculated at the limit concentration. It should be noted that the water solubility of the test item was determined to be <1.5µg/L, which likely explained the high decrease of the concentration during the test period. 

No immobility was observed at any of the test concentrations throughout the test.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EC50 for Daphnia magna exposed to EC 205-363-0 exceeded the solubility limit in test medium, with an average exposure concentration of 0.48 mg/L.