Reaction product with n-butanol of high-boiling stream resulting from the hydroxylation, oxidative cleavage and hydrolysis of vegetable oil saturated and unsaturated C16-C22 triglycerides.

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

October 02, 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

liquid chromatography with MS/MS detection.

Details on sampling:

Sampling: the samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director.
One sample from each solvent stock solution and duplicate samples from the test media of all test concentrations, the solvent control and the control were taken at two points prior to test start to check if the test concentrations have equilibrated in the flow-through system. Then, at test start (day 0) and at regular sampling periods throughout the test at day 7 and day 14, and at test end (21 days) one sample from each solvent stock solution and duplicate samples from the test media of all test concentrations, the solvent control and the control were taken. All biological samples were diluted by a factor of two with acetonitrile. The stock solutions remained undiluted. Additional samples of the control and the dilution solvent acetonitrile were taken at each sampling without any sample treatment.

Storage: all samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and will be kept stored up to the date of the final report.

Vehicle:

yes

Remarks:

DMF (N,N-Dimethylformamide) and Tween 80 (Polysorbate 80)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle1 : DMF (N,N-Dimethylformamide), CAS 68-12-2
- Concentration of vehicle 1 in test medium: 100 µl
- Chemical name of vehicle 2: Tween 80 (Polyoxyethylene (20) sorbitan monooleate), CAS 9005-65-6
- Concentration of vehicle 2 in test medium: 10 µl
- Solvent Control: in the solvent control, test water was used with 100 µl DMF/l and 10 µl Tween 80/l.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: clone 5
- Justification for species other than prescribed by test guideline:
- Source: ibacon's in-house laboratory culture.
- Age: 2 to 19.75 hours old
- Feeding during test: the Daphnia were usually fed with green algae (Desmodesmus subspicatus), freshly grown in the laboratories of ibacon.
- Food type: green algae (Desmodesmus subspicatus)
- Amount:
The amount of food was based on the concentration of total organic carbon (TOC) in the algal food suspension. The feeding would have been adjusted, if a lack of food or an overload would have been observed. The approximate amount of algal TOC/Daphnia per feeding were as follows:
Days 0-3: 0.1 mg
Days 4-7: 0.15 mg
Days 8-21: 0.2 mg
- Frequency: twice a day

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

142.4 to 231.4 CaCO3/l

Test temperature:

19.7 to 20.3 °C

pH:

7.0 to 8.0

Dissolved oxygen:

7.9 to 9.3 mg/l

Nominal and measured concentrations:

NOMINAL: 7, 2.2, 0.7, 0.22 and 0.07 mg test item/l
MEASURED: 6.16, 2.10, 0.507, 0.150 and 0.032 mg test item/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Ten vessels in one basin (1.4 l) per treatment group. Each test unit was uniquely identified with serial number, treatment and replicate number.
- Aeration:
- Type of flow-through: Water exchange: 7.4 fold per day.
- No. of organisms per vessel: 10
- No. of vessels per concentration: 1
- No. of vessels per control : 1
- No. of vessels per vehicle control : 1
This study encompassed 5 test concentrations, a solvent control and a control with 10 replicates, each containing one Daphnia. The Daphnia were exposed for 21 days in a flow through test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Medium
Analytical grade salts and additives will be added at the following nominal concentrations in deionised water:
CaCl2 x 2H2O 2.0 mmol/l (= 294 mg/l)
MgSO4 x 7H2O 0.5 mmol/l (= 123 mg/l)
NaHCO3 0.75 mmol/l (= 65 mg/l)
KCl 0.075 mmol/l (= 5.8 mg/l)
Additionally added will be the Trace Element:
Na2SeO3 2.2 µg/l
And Vitamins:
Thiamin HCl 75.0 µg/l
Cyanocobalamin (B12) 1.0 µg/l
Biotin (B6) 0.75 µg/l
- Alkalinity: 0.9 mmol/l

OTHER TEST CONDITIONS
- Photoperiod:16 h light - 8 h dark
- Light intensity: 1130 to 1500 lux (The light intensity was measured once during the test).

TEST CONCENTRATIONS
- Range finding study: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
The pre-experiments were not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the project number of the present study.
- Test concentrations: 7, 2.2, 0.7, 0.22 and 0.07 mg/l
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Remarks:

Reproduction per Survived Parent

Effect conc.:

0.507 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Cumulative Offspring per Survived Parent

Key result

Duration:

21 d

Dose descriptor:

LOEC

Remarks:

Reproduction per Survived Parent

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Cumulative Offspring per Survived Parent

Key result

Duration:

21 d

Dose descriptor:

EC50

Remarks:

Reproduction per Survived Parent

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Cumulative Offspring per Survived Parent

Remarks on result:

other: 1.51 -3.20 mg test item/l

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 6.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Reported statistics and error estimates:

The mortality of parental daphnia was tested with the Step-down Cochran-Armitage test on a concentration-response pattern.
The difference between the control and the solvent control was tested with a two sample t-test for reproduction and the Fischer´s Exact Test for Mortality.
The NOEC and the LOEC for the reproduction for all introduced daphnia were evaluated by a Step-down Jonckheere-Terpstra test.
The NOEC and the LOEC for the reproduction for all surviving daphnia were evaluated by a Welch-t-test after Bonferroni-Holm.
The NOEC and the LOEC for mortality were evaluated by the Fisher’s exact binominal test.
The EC50, EC20 and EC10 for reproduction were determined by probit analysis.
The EC-value for mortality could not be calculated due to low toxicity of the test item and was determined directly from the raw data.
The statistical evaluation was performed in accordance with the revised version of the OECD Guideline No. 211 from October 02, 2012. Since a Cochran-Armitage trend test revealed no significant trend in mortality, the evaluation was based on the cumulative offspring per survived parent. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

VALIDITY CRITERIA FULFILLED:

- Survival Rate of Adult Daphnia in the Control:  100 % in the Control and 90 % in the Solvent Control; validity criterion was met.

- Mean Number of Live Offspring in the Control after 21 Days: 158 per surviving adult Daphnia in the Control and 88 per surviving adult in the Solvent Control; validity criterion was met.

Mortality of Adults:

The Cochran-Armitage trend test revealed no significant trend in mortality. At the control, the test item concentration of 0.507 and 2.10 mg test item/l, allDaphniasurvived until the end of the test. At the solvent control and the mean measured concentrations of 0.032 and 0.150 mg test item/l oneDaphniadied during the test. At the highest test concentration of 6.16 mg test item/l, threeDaphniadied during the test. There was no significant different to the Solvent Control in mortality of Daphniamagna after 21 days.

Reproduction:

Since the Cochran-Armitage trend test revealed no significant trend in mortality all statistical analyses are based on the number of offspring by all survived parents at test start.

There was a significant difference between the control and the solvent control. Therefore the test concentrations were compared with the solvent control.

The reproduction of the Daphnia was 158 ± 10.6 (mean ± SD) live offspring per surviving adult in the control and88 ± 7.6in the solvent control.

The coefficient of variation around the mean number of living offspring produced per surviving parentDaphniain the control and the solvent control was6.7 %and8.7 %. According to the results of a Welch t-test (one-sided smaller, α= 0.05) no significant toxic effect of the test item on the mean reproduction was determined up to and including the mean measured test concentration of 0.507 mg test item/l. At the concentration of 2.10 mg test item/l, the mean reproduction of surviving Daphnia was statistically significantly reduced to 49.5 % living offspring compared to the solvent control. The EC₅₀of the reproduction was determined to be 2.20 mg test item/l.

Date of First Brood:

At the solvent control first offspring was observed at Day 9.The first offspring released from their parent animals were recorded in the control and the mean measured test concentrations 0.032 and 0.150 mg test item/l at the observation on Day 10. At the concentrations of 0.507 and 6.16 mg test item/l the first offspring was observed on Day 11 and at the concentration of 2.10 mg test item/L on Day 13.

Signs of Intoxication:

Apart from the reported mortality and the reduced reproduction, dead offspring were observed once in the third concentration during the test. According to the Guideline, the dead offspring is not considered in the evaluation of the results.

Summary of Analytical Results

Sample Description [mg/l]	% of nominal	RSD %	Mean measured value mg/l
control	n.a	n.a	n.a
Solvent control	n.a	n.a	n.a
0.07	45	39	0.032
0.22	68	10	0.15
0.7	72	14	0.507
2.2	95	16	2.1
7	88	40	6.16

Validity criteria fulfilled:

yes

Conclusions:

The 21 day NOEC was determined to be 0.507 mg test item/l for the reproduction and ≥ 6.16 mg test item/l for the mortality of the adult test animals. The EC50 for the reproduction was 2.20 mg test item/l.

Executive summary:

The purpose of this study was to evaluate the influence of the test item on the mortality and reproduction of Daphnia magna has been evaluated during an exposure period of 21 days according to the OECD guideline 211 "Daphnia magna Reproduction Test"; Commission Regulation (EC) No 440/2008, Annex, Part C, C.20.: "Daphnia magna Reproduction Test",and SANCO/3029/99 rev.4 11/07/00: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414.

Young Daphnia (< 24 hours old) were exposed in a flow through test to aqueous test media containing the test item at various concentrations.

The nominal concentrations were: 7, 2.2, 0.7, 0.22 and 0.07 mg test item/l (each in 100 µl DMF/L and 10 µl Tween 80/L); corresponding to 6.16, 2.10, 0.507, 0.150 and 0.032 mg test item/l mean measured concentration.

The mortality, number of offspring, and symptoms of intoxication were compared with corresponding parameters in the control.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

The initial concentrations and the maintenance of the exposure concentrations during the test were analysed in the analytical part. All reported results refer to mean measured values since the concentrations of the test item were not within± 20% of the nominal concentrations during the test.

The effects of test item on the mortality and reproduction of Daphnia magna was determined during an exposure period of 21 days. The 21‑day NOEC was determined to be 0.507 mg test item/l for the reproduction and ≥ 6.16 mg test item/l for the mortality of the adult test animals. The EC₅₀ for the reproduction was 2.20 mg test item/l.

Description of key information

The performed tests, present results of doubtful interpretation and a number of confounding factors. Some effects have been reported above the solubility limits and in the presence of co‐solvents that were themselves toxic at the used concentrations; furthermore they had a confounding impact on the surface tension and partition phase of some components of the substance.

The observed effects have been reported much above the solubility limit of most of the components, nevertheless the substance is not Readily Biodegradable and log Kow is higher than 4. As a consequence a precautionary classification as Aquatic Chronic 4 (Safety net) is adopted.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.507 mg/L