D-Glucitol, 1,5-anhydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-, 2,3,4,6-tetrakis(2,2-dimethylpropanoate), (1S)-

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:

13 May 2020 - 18 Jun 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD GD 23 - Guidance Document on aqueous-phase aquatic toxicity testing of difficult test chemicals

Version / remarks:

2019

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number: from sponsor, batch no: I19JB2782
- Purity: 99.6% (w/w), no correction factor required
- Expiration date: 12 October 2021 (retest date)
- Purity test date: 20 October 2019 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility of the test material in the solvent/vehicle and the exposure medium: solubility in vehicle: acetone 150 g/L, water < 0.01 g/L (21.5 °C, pH 7.7), solubility in medium: < 10 µg/L

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10 µg/L and controls
- Sampling method: 1.0 mL samples for analysis were taken from all test concentrations and the controls, from the approximate centre of the solutions. Samples were taken at the start of the test and weekly thereafter from the freshly prepared solutions (day 0, 6 and 14); and at the first renewal and weekly thereafter from the 48-h old solutions (day 2, 8 and 16). At the end of the renewal period, the replicates were pooled at each group before sampling. In addition, reserve samples of 1.0 mL were taken from all test solutions for possible analysis.
- Sample storage conditions before analysis: Test samples not analyzed on the day of sampling were stored in the freezer (≤ -15°C)

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a stock solution of 100 µg/mL in acetone. A volume of 100 µL/L stock was spiked to test medium to obtain a final concentration of 10 µg/L. Subsequently, 15 minutes of magnetic stirring was applied to assure a homogeneous solution. All test solutions were clear and colorless at the end of the preparation procedure
- Controls: blank control and solvent control
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): stock solution: test medium with 100 µg/L acetone in solvent control
- Test concentration separation factor: limit test

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, 1820, at least 3th generation
- Age at study initiation: to initiate the test, young daphnids < 24 hours old were selected
- Method of breeding: Daphnia magna are obtained by cyclical parthenogenesis.
- Source: in-house laboratory culture with known history
- Feeding during test: Twice daily an amount of 0.25 mL of a Chlorella sp. suspension. On weekend days an amount of 0.50 mL was added in one single feed. This daily ration corresponded to 0.2 mg C/Daphnia/day.
- Breeding:
* Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing them individually in 50 mL M7-medium
* Maximum age of the cultures: 4 weeks
* Temperature of medium: 18-22°C
* Medium during breeding: M7
* Feeding frequency during breeding: Daily, a suspension of fresh water algae (Chlorella sp.)
* Validity of the 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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

196-250 mg CaCO3

Test temperature:

19-20°C

pH:

7.2-8.4

Dissolved oxygen:

3.4-9.3 mg O2/L

Nominal and measured concentrations:

Nominal: 10 µg/L
Measured: 8.15 µg/L (day 0; fresh), 3.54 µg/L (day 2; old), 12.3 µg/L (day 6; fresh), 10.4 µg/L day 8; old), 12.8 µg/L (day 14; fresh), 11.9 µg/L (day 16; old)

As some of the measured concentrations deviate by more than +- 20% from the nominal concentrations, the effect parameters were expressed in terms of a Time Weighted Mean (TWM) concentration. This TWM concentration was calculated to be 9.7 µg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass vessel covered with a Perspex plate, volume: 60 mL (6 x Ø 3.5 cm)
- Fill volume: each vessels contains a minimum of 50 mL test medium
- Renewal rate of test solution: every 48 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10 per test group
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no (M7 medium)
- Intervals of water quality measurement:
* Temperature, oxygen and pH: at the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids. Additionally, the temperature was continuously monitored in a temperature-control vessel.
* Hardness: once a week in fresh and old media from the control and the highest test concentration
* TOC: at the start of the test in blank test medium
* Light: at the start and the end of the test

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: daily 16 hours
- Light intensity: The intensity did not exceed 1000-1500 lux which corresponds with a range not exceeding 15-20 µE/m2/s1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Parental daphnids:
* Conditions: Every day, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
* Body length: At the end of the test.
Offspring:
* Appearance first brood, presence of unhatched eggs, incidence of immobility, incidence of entrapment: when observed
* Newborn daphnids: Every day, the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: none (limit test)
- Range finding study: yes
- Test concentrations range finding study: 0.010 - 0.10 - 1.0 - 10 µg/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

Effect conc.:

>= 9.7 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.7 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.7 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

- Mortality of parental daphnids: None of the introduced parental daphnids died during the test period in both control groups and at the limit concentration.
- Reproduction: On average, 127 offspring were produced in the blank control, while 170 in the solvent control. The difference was statistically significant. In the limit concentration the number of offspring was 153 per parental daphnid, i.e. 10% less than in the solvent control. This difference was not statistically significant and therefore, the unbound NOEC for reproduction was thus ≥9.7 μg/L.
- Age at first reproduction: The first brood was observed at day 8 in all test groups.
- Parental body length: No reduction in parental body length was found at the limit concentration when compared to the solvent control.

Reported statistics and error estimates:

- Number of living newborn daphnids per parent: Not performed, since no parental mortality occurred in any test group.
- Reproduction: Shapiro-Wilk´s Test on Normal Distribution, Two-sample test between control and solvent control (STUDENT-t test for Homogeneous Variances), NOEC determination: Two-sample t-test Procedure
- Parental body length: Shapiro-Wilk´s Test on Normal Distribution, STUDENT-t test for Homogeneous Variance, NOEC determination: Two-sample t-test Procedure
- Comparison of controls: STUDENT-t test for Homogeneous Variances (alpha = 0.05, two sided) after checking for normality and homogeneity of variances.

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Mortality (immobility) of parental daphnids at the end of the test

Test item (TWM conc. (µg/L))	Introduced	Mobile	Immobile	% Immobility
Blank control	10	10	0	0
Solvent control	10	10	0	0
9.7	10	10	0	0

Age at first reproduction

Test item (TWM conc. (µg/L))	Mean	Med	Min	Max	n
Blank control	8.9	8.5	8.5	9.5	10
Solvent control	8.9	8.5	8.5	9.5	10
9.7	8.9	8.5	8.5	11.5	10

Cumulative mean number of living offspring per parent and reduction of reproduction at the end of the test

Test item (TWM conc. (µg/L))	Mean	Std. Dev.	n	% reduction
Solvent control	170	26.70	10
9.7	153	23.84	10	10

Incidence of immobilised offspring and aborted eggs

Test item (TWM conc. (µg/L))	Number of
	immobile offspring	aborted eggs
Blank control	0	4
Solvent control	2	3
9.7	2	0

Group mean body length (mm) and reduction of growth of parental daphnids at the end of the test

Test item (TWM conc. (µg/L))	Mean (mm)	Std. Dev.	n	% change
Solvent control	4.47	0.154	10
9.7	4.51	0.161	10	-0.80

 

Validity criteria fulfilled:

yes

Conclusions:

The chronic toxicity of T003422 to Daphnia magna was determined in a 21-day reproduction test according to the OECD guideline 211. The study was carried out as a semi-static limit test, and using acetone as solvent. Under the conditions of the present study, the test item did not affect reproduction, growth and survival of parental Daphnia magna at the TWM concentration of 9.7 µg/L (21-d NOEC >= 9.7 µg/L for reproduction, growth, and mortality). The results of the test can be considered reliable without restriction.

Description of key information

One key study (Tobor-Kaplon, 2020) is used for endpoint coverage. The study investigates the chronic toxicity of T003422 to Daphnia magna in a 21-day reproduction test according to the OECD guideline 211. The study was carried out as a semi-static limit test, and using acetone as solvent. The test item did not affect reproduction, growth and survival of parental Daphnia magna at the TWM concentration of 9.7 µg/L (21-d NOEC >= 9.7 µg/L). The study was assigned a Klimisch score of 1 and was conducted under GLP.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 9.7 µg/L