.alpha.-D-Glucopyranosyl bromide, 2,3,4,6-tetrakis(2,2-dimethylpropanoate)

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 November 2021 - 07 December 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment No. 23 (Second Edition) “Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals.

Version / remarks:

8 Feb 2019

Qualifier:

according to guideline

Guideline:

other: MEP, P.R.China, The Guidelines for the Testing of Chemicals, Effects on Biotic Systems (the Second Edition), No. 210 “Fish, early-life stage toxicity test”. China Environmental Press, 2013.8.

GLP compliance:

no

Remarks:

pending GLP certificate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: M21AB0732
- Purity: 100% (w/w)
- Expiration date: 29 January 2022 (retest date)
- Purity test date: 19 April 2021 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability in water and acetone: not indicated
- Solubility in water: < 0.02 mg/L at 20°C

Analytical monitoring:

yes

Details on sampling:

- Concentrations: determined once a week, in fresh and old media, in the control and all test concentratoins
- Sampling method: 150 mL solution was sampled from each test concentration and control. The pretreatment of test samples was same as the recovery sample
- Sample storage conditions before analysis: the rest was stored in refrigerator (2-8 °C) for possible re-analysis and until the end of the test

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A concentration of 100 mg/L was prepared by adding a weighed amount of 0.20 g (0.2000 – 0.2008 g) test item directly in 2 L test water. After 10 days of test, amount of 0.80 g (0.8001 – 0.8005 g) test item was weighed and added into 8 L test water. The solutions were stirred for 24 hours at approximately 400 rpm to ensure maximum dissolution in test medium. The test solution was filtered through a 0.45 µm filter, to obtain a saturated solution (SS) and used as the highest test concentration. The filtered solution was diluted step-by-step to get the test solutions of 6.25%, 12.5%, 25% and 50% of SS at 100 mg/L
- Controls: A concurrent control group was included in parallel with same volume test water as test group
- Test concentration separation factor: 2

Test organisms (species):

other: Rare Minnow (Gobiocypris rarus)

Details on test organisms:

TEST ORGANISM
- Common name: Rare Minnow
- Source: in-house culture, their parental generation was introduced from CTI Chemical Safety Evaluation Research Center (SUZHOU) CO., LTD
- holding of the brood fish: 16h/8h light/dark, 25°C, oxygen concentration at least 80% of ASV, 2x feeding on weekday, 1x in weekend

POST-HATCH FEEDING
- Start date: 2 days post hatch
- Type/source of feed: brood fish: BSN
- Frequency of feeding: ad libitum whilst minimizing the surplus twice daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

28 days post-hatch

Hardness:

10-250 mg CaCO3/L

Test temperature:

26 +- 1.5°C

pH:

6.0-8.5

Dissolved oxygen:

>60% of air saturation value

Nominal and measured concentrations:

- Nominal concentrations: 6.25%, 12.5%, 25%, 50%, 100% of SS at 100 mg/L
- Measured concentrations (geometric mean of fresh and old media): 0.00676, 0.0164, 0.0296, 0.0601 and 0.134 mg/L. The measured test concentration in the old solution generally remained stable (63%-109% of the fresh solution)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass beakers at the earlier stage of the test and 1000 ml glass beakers after 10 days post-hatch
- Test solution volume: About 200 mL at the earlier stage of the test and 800 mL after 10 days post-hatch
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): renewal every 48h
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Loading rate: 80 fertilised eggs, divided equally between 4 replicate test chambers

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the dilution water of tap water after was treated with Aquapro Pure Water System
- Total organic carbon: < 2 mg/L
- Oxygen concentration and pH values were measured weekly and at the end of the test in all test vessels before and after test solution renewal. Total hardness was measured in the control water at the start and end of the test. Temperature was continuously monitored and recorded by an automatic temperature monitoring system.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Observations on hatching and survival were made one time on 3 hours after exposure and then daily and the number were recorded.
- For dead embryos, larvae and juvenile fish, the abnormal appearance or behaviour observed were also recorded.
- The wet weights on a replicate basis and individual total lengths were measured at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

RANGE-FINDING STUDY (= fish acute test)
- Test concentrations: control and 100% of saturated solution at 100 mg/L (nominal concentration)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.134 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

- Embryonic survival: NOEC = 0.134 mg/L, LOEC > 0.134 mg/L
- Larval survival and development: NOEC = 0.134 mg/L, LOEC > 0.134 mg/L
- Larval growth: NOEC = 0.134 mg/L, LOEC > 0.134 mg/L


Embryonic survival:
- Hatching of the fertilised eggs in the control group and test groups was >90% and completed on day 4. Day 4 was designated as day 0 of the 28 day post-hatch growth period. The mean hatchability of fertilised eggs in the control and nominal concentrations of 6.25%, 12.5%, 25%, 50% and 100% of SS at 100 mg/L was 98.8%, 97.5%, 98.8%, 97.5%, 97.5% and 95.0%, respectively.
- During the exposure period, no abnormal appearance or behavior were observed on fertilised eggs in the control and nominal concentrations of 6.25%, 12.5%, 25%, 50% and 100% of SS at 100 mg/L.

Larval survival and development:
- The mean survival rate of post-hatch was 82.2%, 80.7%, 79.7%, 79.4%, 79.5% and 81.8% in the control and nominal concentrations of 6.25%, 12.5%, 25%, 50% and 100% of SS at 100 mg/L, respectively
- During the exposure period, no abnormal appearance or behavior were observed on juvenile fish in the control and nominal concentrations of 6.25%, 12.5%, 25%, 50% and 100% of SS at 100 mg/L.

Larval growth:
- Mean total length per surviving fish for the control and the test groups was 11.45 to 11.69 mm. The mean wet weight per surviving fish for the control and the test groups were 0.01140 to 0.01253 g.
- There were no statistically significant differences for both body weight and length between the controls and the different test groups (P>0.05).

- The individual results of pH value, dissolved oxygen, water hardness and temperature were in the acceptable range

Reported statistics and error estimates:

For NOEC/LOEC determination, Hatching rate, survival rate of post-hatch success, fish length and wet weight for each test concentration was compared with the control’s using One-way ANOVA and multiple comparison (LSD Test or Dunnet Test) in SPSS (version 28.0).

Summary of measured concentrations

Nominal conc. (% of SS at 100 mg/L)	Measured concentrations (mg/L)										Geometric mean concentration conc. (mg/L)
	0d fresh	2d old	10d fresh	12d old	18d fresh	20d fresh	24d fresh	26d old	28d fresh	30d old
control	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
6.25	0.008	0.006	0.008	0.007	0.006	0.006	0.008	0.007	0.006	0.006	0.007
12.5	0.018	0.014	0.022	0.017	0.015	0.013	0.019	0.018	0.017	0.015	0.016
25	0.035	0.024	0.038	0.026	0.028	0.022	0.033	0.033	0.032	0.028	0.030
50	0.069	0.057	0.083	0.052	0.055	0.039	0.070	0.069	0.062	0.057	0.060
100	0.141	0.154	0.178	0.150	0.122	0.079	0.140	0.142	0.138	0.122	0.134

 

Hatching success

Nominal conc. (% of SS at 100 mg/L)	Initial eggs number	Hatching number	Unhatched number	Mean hatching rate (%)
control	80	79	1	98.8
6.25	80	78	2	97.5
12.5	80	79	1	98.8
25	80	78	2	97.5
50	80	78	0	97.5
100	80	76	4	95.0

 

Larval body length

Nominal conc. (% of SS at 100 mg/L)	Mean total lenght in each test group (mm)	N
control	11.69	65
6.25	11.68	63
12.5	11.62	63
25	11.45	62
50	11.66	62
100	11.57	62

Larval body weight

Nominal conc. (% of SS at 100 mg/L)	Mean wet weight per fish in each test group (g)	N
control	0.012	65
6.25	0.012	63
12.5	0.012	63
25	0.013	62
50	0.012	62
100	0.011	62

Validity criteria fulfilled:

yes

Remarks:

Dissolved oxygen concentration >60% of ASV throughout the test. The water temperature was in the range of 24.5 - 27.4 °C. Overall survival of fertilised eggs in the control groups was 98.8% and survival of post-hatch success was 82.2% in the control.

Conclusions:

A fish early-life stages toxicity test of T003421 was conducted with rare minnow (Gobiocypris rarus) over a 32 days exposure period, according to OECD 210 guideline. Lethal and sub-lethal effects were assessed and compared with control values to determine the various effect concentrations. The overall NOEC of T003421 for the early life stages of fish was 0.134 mg/L. The results of the test can be considered reliable without restriction.

Description of key information

One key study (Hairong, 2022) is used for endpoint coverage. The study investigates the chronic toxicity of T003421 to fish (Gobiocypris rarus) in a 32-day early-life stage toxicity test according to the OECD guideline 210. A 32d-NOEC = 0.134 mg/L was determined based on growth (mean measured concentration). 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.134 mg/L