Lithium bis(oxalato)borate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-07 - 2017-08-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The guidelines for the testing of chemicals (HJ/T 153 -2004 )

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "The Guidelines for the Testing of Chemicals, Effects on Biotoc Systems" (the 2"d edition)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were taken from each concentration during the test at 0, 6, 14, 20 and 28d.
- Sample storage conditions before analysis: The test samples were stored in the freezer (-20°C). The test samples were analysed by ICP-MS until all water samples were collected.

Pre-treatment method of the sample
5.00 mL water samples were collected. Blank controls were directly analysed by ICP-MS. 1.25% parent solutions were diluted to 25.0 mL with 5% nitric acid (5 -folds diluted). 2.5% parent solutions were diluted to 50.0 mL with 5% nitric acid (10-folds diluted).5.0% parent solutions were diluted to 100 mL with 5% nitric acid (20-folds diluted). 10% parent solutions were diluted to 200 mL with 5% nitric acid ( 40-folds diluted). 20% parent solutions were diluted to 400 mL with 5% nitric acid (80-folds diluted). The concentrations of test substance were calculated by the ICP-MS response of Li.

Vehicle:

yes

Remarks:

water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Good quality tap water which had been dechlorinated for at least 24 h was used.
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): tap water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Stock solution with a nominal concentration of 100 mg/L was prepared by adding about 0.30 g
test substance to 3 L test medium. The mixture was magnetically stirred for 30 minutes and the test item is fully dissolved. The pH was adjusted to approximately 8.5 using 0.1 M Sodium hydroxide and the dispersions were filtered through a rough paper filter (-5 μm) to remove any precipitate. The resulting Stock Solutions (SS) which were clear and colourless were used as highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS. The test solutions were renewed every two days during the study period.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- Common name: Chinese rare minnow
- Batch No.: JF20160925G
- Source: own lab brood
- Age at study initiation (mean and range, SD): 0.0512 g (0.137 - 1.62 g)
- Method of breeding: own lab
- Feeding during test : The fish were fed daily with live food (Artemia) daily and the quantity of food being kept constant at 4% dry weight related to the initial fi sh weight. Food was withheld from the stock population for 24 h prior to the start of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

145 mg (CaCO3)/L

Test temperature:

23.1°C to 23.5°C

pH:

7.2

Dissolved oxygen:

90%-95%, Not less than 60 per cent of the maximum air saturation value throughout the test; aeration forbidden.

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

nominal: 1.25, 2.50, 5.00, 10.0 and 20.0 mg/L
measured: 1.35, 2.50, 4.97, 9.92 and 19.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: tanks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Made of chemically inert material, with a sealable inert lid, and with a capacity of approximately 5 L.
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): every 48 hours
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 0.2 to 0.4 g of fish/L

TEST MEDIUM / WATER PARAMETERS
No Items Analyzed value Acceptable value*
1 As < 0.001 mg/L ≤ 0.001 mg/L
2 Cd <0.0001 mg/L ≤ 0.0001 mg/L
3 Pb < 0.001 mg/L ≤ 0.001 mg/L
4 Hg < 0.0001 mg/L ≤ 0.001 mg/L
5 Co < 0.001 mg/L ≤ 0.001 mg/L
6 Ni < 0.0002 mg/L ≤ 0.0005 mg/L
7 Cu < 0.01 mg/L ≤ 0.01 mg/L
8 Cr6+ < 0.004 mg/L ≤0.01 mg/L
9 Fe < 0.01 mg/L ≤ 0.3 mg/L
10 Zn 0.01 mg/L ≤ 1.0 mg/L
11 Ag < 0.001 mg/L ≤ 0.05 mg/L
12 pH 7.20 6.0 - 8.5
13 The total hardness 145 mg (CaCO3)/L 10 - 250 mg (CaC03)/L
14 Visible substance NA ≤ 5 mg/L
15 Malathion < 0.001 mg/L ≤ 0.005 mg/L
16 Methyl parathion < 0.001 mg/L ≤ 0.002 mg/L
17 Parathion < 0.001 mg/L ≤ 0.003 mg/L
18 Total organic carbon
(TOC) < 0.20 mg/L ≤ 2 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: 16-hour light and 8-hour dark cycle daily.
- Temp.: 22.9°C - 23 .2°C

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- observations: The fish was examined daily during the test period and any external abnormalities (such as hemorrhage, discoloration) and abnormal behaviour (such as inactivity, strange swimming pattern, other abnormal behaviour, etc.) were noted.
Fish were considered dead if there was no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction. The dead fish were removed as soon as possible, if any.
- Measurements: At the start and the end of the test, all surviving fish were weighed as wet weights (blotted dry) by individual.
Each freshly prepared treatment (day 0) and each old test medium (day 6, 14, 20, 28) were analysed.
Measurements of pH, dissolved oxygen and temperature were carried out at the beginning and thereafter (0, 6, 14, 20, 28 d).

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
According to the report of the acute toxicity of test item (LIBOB PREMIUM BATTERY GRADE) to zebrafish (Brachydanio rerio) (Report No: R2010NC027), the LOEC and 96 h-LC50 of the test item to zebrafish were all greater than 150 mg/L (measured concentration 139 mg/L).
So the concentration of 1.25, 2.50, 5.00, 10.0 and 20.0 mg/L were assigned in the test. Synchronously blank control with no test substance was used in the test.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Fish weights (individual and mean values) on day 0, 14 (if measured) and 28: see Table [1]
- Tank-average or pseudo-specific growth rates for periods 0-28: see Table [2]
- Type of and number with morphological abnormalities: 0
- Type of and number with behavioural abnormalities: 0
- Other biological observations: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Incidents in the course of the test which might have influenced the results: none

Reported statistics and error estimates:

One-Way ANOVA Analysis of the Pseudo Specific Growth Rates
Total: Mean 5.446416, St. Dev. 0.08958386, Freq. 60

Table 1 Individual and Mean Fish Weights on Day 0

No.	Nominal Concentration( mg/L)
	Blank control	1.25	2.50	5.00	10.0	20.0
1	0.0506	0.0514	0.0516	0.0518	0.0520	0.0518
2	0.0508	0.0516	0.0508	0.0514	0.0516	0.0520
3	0.0513	0.0518	0.0511	0.0516	0.0514	0.0514
4	0.0514	0.0516	0.0516	0.0512	0.0518	0.0516
5	0.0510	0.0511	0.0515	0.0517	0.0516	0.0517
6	0.0514	0.0514	0.0514	0.0520	0.0510	0.0521
7	0.0511	0.0512	0.0513	0.0519	0.0515	0.0511
8	0.0520	0.0521	0.0512	0.0523	0.0516	0.0515
9	0.0508	0.0516	0.0516	0.0516	0.0515	0.0513
10	0.0513	0.0520	0.0518	0.0515	0.0513	0.0516
Mean (g)	0.0512	0.0516	0.0514	0.0517	0.0515	0.0516
Range of body weight (%)	0.137~1.62	0.039~1.01	0.019~1.150	0~1.16	0.058~1.03	0.019~0.988

*：(Body weight-Mean weight)/Mean weight×100%.

Table 2 Individual and Mean Fish Weights on Day 28

No.	Nominal Concentration (mg/L)
	Blank control		1.25		2.50		5.00		10.0		20.0
	Weights	Rates^1	Weights	Rates	Weights	Rates	Weights	Rates	Weights	Rates	Weights	Rates
	(g)	(%)	(g)	(%)	(g)	(%)	(g)	(%)	(g)	(%)	(g)	(%)
1	0.2318	5.39	0.2379	5.46	0.2345	5.42	0.2367	5.43	0.2288	5.326	0.2327	5.38
2	0.2329	5.41	0.2377	5.46	0.2391	5.49	0.2359	5.42	0.2339	5.405	0.2387	5.47
3	0.2492	5.65	0.2345	5.41	0.2353	5.43	0.2379	5.45	0.2275	5.306	0.2708	5.92
4	0.2425	5.55	0.2372	5.45	0.2332	5.40	0.2357	5.42	0.2292	5.332	0.2335	5.39
5	0.2372	5.48	0.2361	5.43	0.2355	5.44	0.2367	5.43	0.2372	5.455	0.2391	5.48
6	0.2335	5.42	0.2372	5.45	0.2378	5.47	0.2332	5.38	0.2292	5.332	0.2337	5.39
7	0.2442	5.58	0.2393	5.48	0.2364	5.45	0.2347	5.40	0.2294	5.335	0.2324	5.37
8	0.2288	5.35	0.2362	5.43	0.2359	5.44	0.233	5.38	0.2389	5.480	0.2426	5.53
9	0.2389	5.50	0.2363	5.43	0.2352	5.43	0.2486	5.61	0.2372	5.455	0.2368	5.44
10	0.2377	5.48	0.2345	5.41	0.2378	5.47	0.2434	5.53	0.2368	5.449	0.2360	5.43
LOEC			> 20.0 mg/L (measured concentration was 19.6 mg/L)
NOEC			= 20.0 mg/L (measured concentration was 19.6 mg/L)

¹Rates:pseudo specific growth ratesfor the periods from day 0 to day 28

Stability of Test Substance in Test Solutions

The analytical results showed that the test solutions with nominal concentrations of 1.25, 2.50, 5.00, 10.0 and 20.0 mg/L, the mean measured concentrations were 1.35, 2.50, 4.97, 9.92 and

19.6 mg/L, respectively. This indicated that concentration of test substance was stable (within 80% of the initial concentration) in the water during the test period. Thus semi-static conditions

of 48 h-renewal used in the test was reasonable.

Test Condition

During the whole test period, the pH values of the control mediums and test media were between 7.66 and 7.89, and the Dissolved Oxygen (DO) values varied from 92.7% ~ 96.9% of the air

saturation at the test temperature, and the temperature of the test media was maintained in 22.9°C ~23.2°C.

Validity criteria fulfilled:

yes

Conclusions:

The results showed that under valid semi-static test conditions ( 48h-renewal), in the 28 d test period, the LOEC (lowest observed effect concentration) and the NOEC (No observed effect concentration) for the growth rate of the test substance to rare minnow were concluded as follows:
28 d-LOEC > 20 mg/L (measured concentration was 19.6mg/L);
28 d-NOEC = 20 mg/L (measured concentration was 19.6 mg/L).

Executive summary:

Under semi-static conditions of 48h-renewal, the effect on growth rates and other observed effects in juvenile fish (Gobiocypris rarus) exposed to the test substance for 28 days was conducted according to the following guidelines: "The guidelines for the testing of chemicals" (HJ/T 153 -2004 ) and the OECD Guideline 215 "Fish, Juvenile Growth Test" (2000) and in compliance with GLP criteria.

Nominal concentrations of 0, 1.25%, 2.5%, 5%, 10% and 20% parent solutions were used in the test.

During the whole test period, the pH values of the control and test media were between 7.66 and 7.89, and the Dissolved Oxygen (DO) values varied from 92.7% ~ 96.9% of the air saturation at the test temperature, and the temperature of the test media was maintained at 22.9 °C ~ 23.2 °C.

All fishes in the control group were normal. The mean weight of fish in the control group increased more than 50%. So the study met the acceptability criteria prescribed by the protocol (dissolved oxygen concentration: no less than 60% of the air saturation value; temperature: (23±2) °C and not differ by more than ± 1°C between test chambers; the increasing rate of the mean wet-weight of fish: no less than 50% of the initial weight). Therefore the test is valid.

ICP-MS was used to determine to the concentration of the test substance in the test medium based on the Lithium ion. For the test solutions with the concentration of 1.25, 2.50, 5.00, 10.0 and 20.0 mg/L, the mean measured concentrations were 1.35, 2.50, 4.97, 9.92 and 19.6 mg/L, respectively. This indicated that the test concentration for the test solution (based on the lithium ion) was stable throughout a period of 48h (deviation within 20%). Thus a semi-static test of 48h-renewal was reasonable.

The test results showed that under valid semi-static test conditions ( 48h-renewal), all fish in the control groups and treated groups (1.25, 2.50, 5.00, 10.0 and 20.0 mg/L) were alive and appeared normal. In the 28d test period, the LOEC of the test substance for the juvenile fish growth rate is greater than 20.0 mg/L (measured concentration was 19.6 mg/L), and NOEC is equal to20.0 mg/L (measured concentration was 19.6 mg/L), thus:

28 d-LOEC > 20.0 mg/L (measured concentration was 19.6 mg/L);

28 d-NOEC = 20.0 mg/L (measured concentration was 19.6 mg/L).

Description of key information

Lithium bis(oxalato)borate did not reveal chronic toxicity to Gobiocypris rarus in a semi-static test resulting in a 28d NOEC for growth rate of 20 mg/L. The corresponding LOEC is > 20 mg/L, respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

20 mg/L

Additional information

Under semi-static conditions of 48h-renewal, the effect on growth rates and other observed effects in juvenile fish (Gobiocypris rarus) exposed to the test substance for 28 days was conducted according to the following guidelines: "The guidelines for the testing of chemicals" (HJ/T 153 -2004 ) and the OECD Guideline 215 "Fish, Juvenile Growth Test" (2000) and in compliance with GLP criteria.

Nominal concentrations of 0, 1.25%, 2.5%, 5%, 10% and 20% parent solutions were used in the test.

During the whole test period, the pH values of the control and test media were between 7.66 and 7.89, and the Dissolved Oxygen (DO) values varied from 92.7% ~ 96.9% of the air saturation at the test temperature, and the temperature of the test media was maintained at 22.9 °C ~ 23.2 °C.

All fishes in the control group were normal. The mean weight of fish in the control group increased more than 50%. So the study met the acceptability criteria prescribed by the protocol (dissolved oxygen concentration: no less than 60% of the air saturation value; temperature: (23±2) °C and not differ by more than ± 1°C between test chambers; the increasing rate of the mean wet-weight of fish: no less than 50% of the initial weight). Therefore the test is valid.

ICP-MS was used to determine to the concentration of the test substance in the test medium based on the Lithium ion. For the test solutions with the concentration of 1.25, 2.50, 5.00, 10.0 and 20.0 mg/L, the mean measured concentrations were 1.35, 2.50, 4.97, 9.92 and 19.6 mg/L, respectively. This indicated that the test concentration for the test solution (based on the lithium ion) was stable throughout a period of 48h (deviation within 20%). Thus a semi-static test of 48h-renewal was reasonable.

The test results showed that under valid semi-static test conditions ( 48h-renewal), all fish in the control groups and treated groups (1.25, 2.50, 5.00, 10.0 and 20.0 mg/L) were alive and appeared normal. In the 28d test period, the LOEC of the test substance for the juvenile fish growth rate is greater than 20.0 mg/L (measured concentration was 19.6 mg/L), and NOEC is equal to 20.0 mg/L (measured concentration was 19.6 mg/L), thus:

28 d-LOEC > 20.0 mg/L (measured concentration was 19.6 mg/L);

28 d-NOEC = 20.0 mg/L (measured concentration was 19.6 mg/L).