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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-03 until 2011-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
, adopted July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method and sample storage conditions before analysis:
For the analysis of the test item concentrations and the control, samples were taken weekly on day 0, 7, 14, 21, 28 and 34. Samples of the test solutions were taken from the freshly prepared test solutions and from the "old" test solutions immediately prior to the renewal of the test solution. The representative samples were taken in duplicate series from each test concentration and control. One series of samples was analyzed immediately after sampling and the second series of samples was stored frozen (-20°C) immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
The test item was formulated in the dilution water without use of any solubilising agent. The pH value of treatment solution was measured and adjusted to that of the dilution water with 1N HCl. This pH adjustment was made in such a way that the solution concentration did not change to any significant extent and that no chemical reaction or physical precipitation of the test substance was caused. Treatment solutions were prepared freshly before the first treatments and renewals. Once the solutions prepared the appropriate amounts were used and the remaining test solutions were stored under ambient temperature and used as necessary within the stability period.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Source: The following breeding stocks were used for production of eggs:
1. H/BR/G/2011-01-26 obtained from Department of Aquaculture, Szent István University (H-2103 Gödöllő, Páter K. u. 1, Hungary) and held in Fish Laboratory of Toxi-Coop Zrt. from 26 January 2011
2. H/BR/TC/2011-03-03 bred in Fish Laboratory of Toxi-Coop Zrt. This stock was established and held in the laboratory from 03 March 2011.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs and subsequent handling of eggs:
Appropriate numbers of fertilized eggs were placed (using a pipette) into beakers containing the appropriate test solutions (test item or control) for pre-treatment of the eggs. The end of the pipette tip was cut to a suitable size compatible with the size of eggs. In this way exposition with the test item could be started as early as possible at the stage of embryonic development. Per test concentration examined, 80 or 79 eggs were planned. As for every test concentration examined and control, 2 replicates were prepared, 40 eggs were transferred to each replicate dish of one concentration level or 40 eggs and 39 eggs for each replicate of another concentration level. The dishes were placed into the incubator. Test organisms were kept in the incubator until the test animals in the test chamber became free-feeding (~ day 5, when the yolk sac of the larvae was eliminated).

POST-HATCH FEEDING
- Start date, type/source of feed, amount given and frequency of feeding:
The fish were fed from elimination of yolk sac (free-feeding stage) to the end of the test twice a day except on the first feeding day (Day 5) when food was supplied once only (dry food was introduced in the afternoon) with appropriate type and amount of food. Dry food (small granule) was used from Day 5 to Day 27.
Dry food (DF) was weight and suspended in the dilution water at each occasion. From Day 5 to Day 20 0.875 g dry food was suspended in 14 mL dilution water and 1 mL of this suspension was supplied for each test aquarium. This procedure was repeated twice daily (except on Day 5). From Day 21 to Day 27 0.438 g dry food was suspended in 14 mL dilution water and 1 mL of this suspension was supplied for each test aquarium per each occasion. This procedure was repeated twice daily.
Live food (Artemia nauplii; AN) was introduced on Day 14. From Day 14 to Day 27 both type of food (DF and AN) was supplied to the animals. From Day 28 only live food was supplied. For producing live food brine shrimp eggs (approximately 1.5 mL from Day 14 to Day 30, and approx. 3 mL from Day 31 to Day 33) were placed into salt water (12.5 g NaCl/L dilution water, 500 mL) and left to hatch for approximately 24 hours. Hatched nauplies were filtered and suspended in 14 mL dilution water and supplied in 0.5 mL aliquots to each test aquarium. The remaining nauplies were placed back into the salt water (same as used for hatching) and kept on the temperature controlled tray until the second feeding of the day. Than filtered and suspended again and supplied in 0.5 mL aliquots to each test aquarium.
Using the above written procedure both type of food were supplied ad libitum during the whole feeding period.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
34 d
Remarks on exposure duration:
30 days after hatching of all control fish.
Post exposure observation period:
No
Hardness:
Total hardness: 178 - 237 mg CaCO3/ L
Test temperature:
24.4 - 25.6 °C
pH:
7.15 - 7.84
Dissolved oxygen:
61.2 % - 97.2 %
Nominal and measured concentrations:
Nominal concentrations: 1.8, 3.0, 7.2, 15, 21.1 mg/L
Mean measured concentrations: 2.43, 3.82, 8.60, 17.35, 24.35 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Suitable-sized petri dishes with 50 mL test solutions was used until free-feeding stage (from day 0 to day 5); suitable sized glass aquaria with 500/ 1000 mL test solution was used from free-feeding stage to the end of the test (from day 5 to day 34)
- Aeration: Test solutions were not aerated until start of feeding. After start of feeding of the larvae the test solutions were slightly aerated until end of the test.
- Renewal rate of test solution (frequency): weekly
- No. of fertilized eggs/embryos per vessel: approximately 40
- No. of vessels per concentration: 2
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (ISO medium, prepared according to recommendation of Annex 2 of the OECD 203 guideline) diluted with purified water (in a ratio of 3 : 2 = reconstituted water: purified water) was used as dilution water in the test. The content of the dilution water was as follows (nominal concentration in the dilution water (reconstituted water: purified water = 3:2 mg/L)
CaCl2 x H2O = 176.4 mg/L
MgSO4 x H2O = 73.8 mg/L
NaHCO3 = 39 mg/L
KCl = 3.48 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: pH value of treatment solutions was measured and adjusted to that of the dilution water with 1N HCl. This pH adjustment was made in such a way that the solution concentration did not change to any significant extent and that no chemical reaction or physical precipitation of the test substance was caused.
- Photoperiod: 16 h light: 8 h dark

EFFECT PARAMETERS MEASURED:
Test organisms were observed in the test chambers for abnormal appearance and behavior as much as possible. At the embryonic and larvae stage microscopic observations were made. After hatching and transfer of larvae form petri dishes to a larger volume test aquariums observations were made either by the naked eye or with a magnifier. The following observations were recorded during the test:
- mortality, each day
- number of healthy fish at the end of the test
- time of hatching start and end of hatching
- numbers of larvae hatching each day
- length (individual) and weight (group) of surviving animals (Due to small size of fish body weights of the treatment groups were measured. Dry weights were determined on day 35 after drying of fish for 24 hours on 60 °C)
- numbers of deformed embryo/larvae/fish (when observed) (abnormal animals were removed from the test chambers only on death)
- numbers of fish exhibiting abnormal behavior (when observed) (uncoordinated swimming, atypical quiescence or atypical feeding behavior).

VEHICLE CONTROL PERFORMED:
The dilution water (without addition of the test item) was used as a control.

RANGE-FINDING STUDY
- Test concentrations: 25 mg/L (nominal)
- Results used to determine the conditions for the definitive study: NOEC (14 d) >/= 25 mg/L
Reference substance (positive control):
no
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
24.35 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
LiOH*H2O
Basis for effect:
mortality
Remarks on result:
other: The observed effect (mortality) was associated with larval/ juvenile stages but not at the embryonic stage.
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
17.35 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
LiOH*H2O
Basis for effect:
mortality
Remarks on result:
other: The observed effect (mortality) was associated with larval/ juvenile stages but not at the embryonic stage.
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
40 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: calculated for lithium nitrate
Basis for effect:
mortality
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: calculated for lithium nitrate
Basis for effect:
mortality
Details on results:
Cumulative mortality: statistically significant (p < 0.01) cumulative mortality compared to the control was observed at treatment concentration of 24.35 mg/L (21.1 mg/L nominal). No significant lethal effect was observed until embryonic/eleutheroembryonic stage. Since mortality observed from Day 6 to Day 34 was statistically significant (p < 0.01) it is considered that the test item influenced the survival of the test organism at larval/juvenile stage rather than at embryonic stage. No statistically significant lethal effect was observed in other test concentrations (neither during embryonic nor larval/juvenile stage).

No significant effect on hatching of the larvae was observed at any concentrations tested. No significant differences were observed either in starting or duration of hatching. Numbers of larvae hatched daily were statistically evaluated on Day 4 and Day 5 and no significant differences were observed.

- Number of eggs treated: control - 40 / 40, 2.43 mg/L dose group - 40/ 42, 3.82 dose group - 40/ 40, 8.60 mg/L dose group - 40 / 41, 17.35 mg/L dose group - 39 / 40, 24.35 mg/L - 40 / 40
- Total number of larvae hatched: control - 37 / 35, 2.43 mg/L dose group - 38/ 37, 3.82 dose group - 38/ 36, 8.60 mg/L dose group - 39 / 39, 17.35 mg/L dose group - 35 /38 24.35 mg/L - 36 / 38
- Number of living fish at the end of the test: control - 34 / 32, 2.43 mg/L dose group - 30/ 33, 3.82 dose group - 31/ 32, 8.60 mg/L dose group - 32 / 37, 17.35 mg/L dose group - 30 /34, 24.35 mg/L - 21 / 23
- Total number of dead animals: control - 6 / 8, 2.43 mg/L dose group - 10/ 9, 3.82 dose group - 9/ 8, 8.60 mg/L dose group - 8 / 4, 17.35 mg/L dose group - 9 / 6, 24.35 mg/L - 21 / 23
- Hatching success (%) mean: control - 90, 2.43 mg/L dose group - 91.5, 3.82 dose group - 92.5, 8.60 mg/L dose group - 96.3, 17.35 mg/L dose group - 92.4, 24.35 mg/L - 92.5
- Post hatch success (%) mean: control - 91.7, 2.43 mg/L dose group - 84.1, 3.82 dose group - 85.2, 8.60 mg/L dose group - 88.5, 17.35 mg/L dose group - 87.6, 24.35 mg/L - 59.4
Overall survival (%) mean: control - 82.5, 2.43 mg/L dose group - 76.8, 3.82 dose group - 78.8, 8.60 mg/L dose group - 85.1, 17.35 mg/L dose group - 81.0, 24.35 mg/L - 55.0
- Cumulative mortality (%) mean - 17.5, 2.43 mg/L dose group - 23.2, 3.82 dose group - 21.2, 8.60 mg/L dose group - 14.9, 17.35 mg/L dose group - 19.0, 24.35 mg/L - 45.0

At the end of the test, group body weights were measured. No significant differences were observed.
Body length was measured for each animal individually and the data were statistically evaluated. Statistically significant (p < 0.05), but biologically not relevant differences were observed at test concentration of 8.60 mg/L (7.2 mg/L nominal).

Other sub-lethal effects were monitored with limited extent: Numbers of embryos/larvae/juveniles with deformations or abnormal behavior were recorded. No obvious test item related changes in the behavior of fish could be observed.

The observed abnormalities recorded during the embryonic stage were mainly gross body deformities (deformed spine). In larval stage inappropriately filled up swim-bladder was observed as a consequence of abnormal development in embryonic stage. No obviously treatment related symptoms were observed. The nature and frequency of these abnormalities in the test item treated groups did not differ from the control. Abnormally developing animals were not able to swim (decreased activity was observed) and feed appropriately, hence died within few days. Neither of the animals with observed symptoms survived the test, since no fish were observed with abnormal appearance or behavior at the end of the test.
Results with reference substance (positive control):
No positive control was used
Reported statistics and error estimates:
Data management and calculation of basic statistics were made with Microsoft Excel Software. The statistical evaluation of data (body length, mortality and hatching rates) was performed with the program package SPSS PC + 4.0.1.
For statistical evaluation of body length data the homogeneity of variance between groups was checked with Bartlett’s homogeneity of variance test. Since significant heterogeneity was found at Bartlett’s test, the non-parametric Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Mortality and hatching rates data were statistically evaluated by Chi-square test.
Validity criteria fulfilled:
yes
Conclusions:
Under conditions of the study, a LOEC value of 24.35 mg test item/L and a NOEC value of 17.35 mg test item/L were calculated for lithium hydroxide monohydrate.
Executive summary:

A chronic toxicity study on fish with lithium nitrate is not available. Consequently, read-across was applied using a characteristically similar compound, lithium hydroxide.

The purpose of this study was to evaluate the chronic toxicity of the test item lithium hydroxide monohydrate to early life stages (embryo, larvae and juveniles) of fish.

For this purpose, eggs were exposed in a semi-static test to aqueous test media containing the test item for 34 days at a range of concentrations under defined conditions. Lethal and sub-lethal effects were assessed and compared with control values to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC) for each response assessed.

The test was considered to be valid as assay acceptance criteria were fulfilled.

Based on the preliminary chronic fish toxicity test result the following nominal test concentrations were selected for the early life stage toxicity test: 1.8 mg/L, 3.0 mg/L, 7.2 mg/L, 15.0 mg/L and 21.1 mg/L.

Test item concentrations were analyzed in the test solutions by flame photometry. Lithium hydroxide monohydrate concentration in the test solutions was determined at 5 renewal periods by flame photometry. The measured concentrations of the lithium hydroxide monohydrate varied between 102 % and 151 % of the nominal concentration during the test. Mean measured concentrations were calculated for each parallel test chambers and for each treatment concentrations. The measured concentrations of each parallel treatment were within the range of ± 20 % of the mean measured values meeting the validity criteria. Since concentrations of the test substance were satisfactorily maintained within ± 20 % of the mean measured values during the test in all treatment groups, all biological results are reported on the basis of the mean measured concentrations: 2.43 mg/L, 3.82 mg/L, 8.60 mg/L, 17.35 mg/L and 24.35 mg/L.

79 to 82 eggs were tested per test concentration and the control in two parallels in the test groups and in the control group (approximately 40-40 eggs per each parallel treatments). Based on microscopic observation, embryos were at approximately 64 to 128 cell stages at start of treatment.

Environmental parameters (water temperature, pH, LDO) were monitored during the test. There were no deviations from the defined ranges.

Animals were fed from elimination of the yolk sac (free-feeding stage) to the end of the test with appropriate type and amount of food ad libitum, and thus starvation did not have any effect on the results obtained.

Cumulative mortality (observed from Day 0 to the end of the test on Day 34), mortality observed at embryonic/eleutheroembryonic (i.e. early larval) stage (from Day 0 to the end of hatching on Day 5) and mortality observed at larval/juvenile stage (from Day 6 - free feeding stage - to the end of the test on Day 34) were statistically evaluated. Statistically significant (p < 0.01) cumulative mortality compared to the control was observed at treatment concentration of 24.35 mg/L (21.1 mg/L nominal). No significant lethal effect was observed until embryonic/eleutheroembryonic stage. Since mortality observed from Day 6 to Day 34 was statistically significant (p < 0.01) it is considered that the test item influenced the survival of the test organisms at larval/juvenile stage rather than at embryonic stage. No statistically significant lethal effect was observed in other test concentrations (neither during embryonic nor larval/juvenile stage).

No significant effect on hatching of the larvae was observed at any concentrations tested. No significant differences were observed either in starting or duration of hatching. Numbers of larvae hatched daily were statistically evaluated on Day 4 and Day 5 and no significant differences were observed.

At the end of the test, group body weights were measured. No significant differences were observed.

Body length was measured for each animal individually and the data were statistically evaluated. Statistically significant (p < 0.05), but biologically not relevant differences were observed at test concentration of 8.61 mg/L (7.2 mg/L nominal).

Other sub-lethal effects were monitored with limited extent: numbers of embryos/larvae/juveniles with deformities or abnormal behavior were recorded. No obvious test item related changes in the behavior of fish could be observed.

Under the conditions of this early life stage toxicity study, the test item lithium hydroxide monohydrate had significant lethal effect on early life stages of Zebrafish (Danio rerio). The observed effect was associated with larval/juvenile stages, but no significant effect was observed during the embryonic stage.

The following endpoints (34 days LOEC and NOEC) were calculated in the study:

The 34 d LOEC 24.35 mg test item/L

The 34 d NOEC 17.35 mg test item/L

Endpoint:
long-term toxicity to fish, other
Remarks:
legislative text
Type of information:
other: legislative text
Adequacy of study:
supporting study
Study period:
1991, 1998, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Legal Directives of the European Community regarding nitrate pollution in drinking water.
Principles of method if other than guideline:
Legislative text. No guideline indicated.
Key result
Dose descriptor:
other: limit value
Effect conc.:
50 mg/L
Conc. based on:
other: nitrate ions
Basis for effect:
not specified
Conclusions:
A limit concentration of 50 mg/L nitrate (equivalent to 55.6 mg LiNO3/L) in drinking water was agreed in the Drinking water Directive.
Executive summary:

As laid down in Council Directive 98/83/EC a limit concentration of 50 mg/L nitrate (equivalent to 55.6 mg LiNO3/L) in drinking water was agreed. Also Directive 2000/60/EC and Council Directive 91/676/EEC refer to the limit value laid down in the Drinking Water Directive.

Endpoint:
long-term toxicity to fish, other
Remarks:
published data
Type of information:
other: published data
Adequacy of study:
supporting study
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Key result
Dose descriptor:
other: toxicity limit
Effect conc.:
ca. 100 - ca. 300 mg/L
Conc. based on:
not specified
Basis for effect:
not specified
Conclusions:
According to the published data the toxicity limit for nitrate ions towards fish is around 100-300 mg/L.
Executive summary:

According to the published data the toxicity limit for nitrate ions towards fish is around 100-300 mg/L.

Description of key information

Under conditions of the study, a LOEC value of 24.35 mg test item/L and a NOEC value of 17.35 mg test item/L were calculated for lithium hydroxide monohydrate. Based on read-across approach, the calculated LOEC and NOEC values for lithium nitrate were 40 and 28 mg/L, respectively.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
28 mg/L

Additional information

Key study

A chronic toxicity study on fish with lithium nitrate is not available. Consequently, read-across was applied using a characteristically similar compound, lithium hydroxide monohydrate.

The purpose of this study was to evaluate the chronic toxicity of the test item lithium hydroxide monohydrate to early life stages (embryo, larvae and juveniles) of fish.

For this purpose, eggs were exposed in a semi-static test to aqueous test media containing the test item for 34 days at a range of concentrations under defined conditions. Lethal and sub-lethal effects were assessed and compared with control values to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC) for each response assessed.

The test was considered to be valid as assay acceptance criteria were fulfilled.

Based on the preliminary chronic fish toxicity test result the following nominal test concentrations were selected for the early life stage toxicity test: 1.8 mg/L, 3.0 mg/L, 7.2 mg/L, 15.0 mg/L and 21.1 mg/L.

Test item concentrations were analyzed in the test solutions by flame photometry. Lithium hydroxide monohydrate concentration in the test solutions was determined at 5 renewal periods by flame photometry. The measured concentrations of the lithium hydroxide monohydrate varied between 102 % and 151 % of the nominal concentration during the test. Mean measured concentrations were calculated for each parallel test chambers and for each treatment concentrations. The measured concentrations of each parallel treatment were within the range of ± 20 % of the mean measured values meeting the validity criteria. Since concentrations of the test substance were satisfactorily maintained within ± 20 % of the mean measured values during the test in all treatment groups, all biological results are reported on the basis of the mean measured concentrations: 2.43 mg/L, 3.82 mg/L, 8.60 mg/L, 17.35 mg/L and 24.35 mg/L.

79 to 82 eggs were tested per test concentration and the control in two parallels in the test groups and in the control group (approximately 40-40 eggs per each parallel treatments). Based on microscopic observation, embryos were at approximately 64 to 128 cell stages at start of treatment.

Environmental parameters (water temperature, pH, LDO) were monitored during the test. There were no deviations from the defined ranges.

Animals were fed from elimination of the yolk sac (free-feeding stage) to the end of the test with appropriate type and amount of food ad libitum, and thus starvation did not have any effect on the results obtained.

Cumulative mortality (observed from Day 0 to the end of the test on Day 34), mortality observed at embryonic/eleutheroembryonic (i.e. early larval) stage (from Day 0 to the end of hatching on Day 5) and mortality observed at larval/juvenile stage (from Day 6 - free feeding stage - to the end of the test on Day 34) were statistically evaluated. Statistically significant (p < 0.01) cumulative mortality compared to the control was observed at treatment concentration of 24.35 mg/L (21.1 mg/L nominal). No significant lethal effect was observed until embryonic/eleutheroembryonic stage. Since mortality observed from Day 6 to Day 34 was statistically significant (p < 0.01) it is considered that the test item influenced the survival of the test organisms at larval/juvenile stage rather than at embryonic stage. No statistically significant lethal effect was observed in other test concentrations (neither during embryonic nor larval/juvenile stage).

No significant effect on hatching of the larvae was observed at any concentrations tested. No significant differences were observed either in starting or duration of hatching. Numbers of larvae hatched daily were statistically evaluated on Day 4 and Day 5 and no significant differences were observed.

At the end of the test, group body weights were measured. No significant differences were observed.

Body length was measured for each animal individually and the data were statistically evaluated. Statistically significant (p < 0.05), but biologically not relevant differences were observed at test concentration of 8.61 mg/L (7.2 mg/L nominal).

Other sub-lethal effects were monitored with limited extent: numbers of embryos/larvae/juveniles with deformities or abnormal behavior were recorded. No obvious test item related changes in the behavior of fish could be observed.

Under the conditions of this early life stage toxicity study, the test item lithium hydroxide monohydrate had significant lethal effect on early life stages of Zebrafish (Danio rerio). The observed effect was associated with larval/juvenile stages, but no significant effect was observed during the embryonic stage.

The following endpoints (34 days LOEC and NOEC of lithium hydroxide monohydrate) were calculated in the study:

The 34 d LOEC 24.35 mg test item/L

The 34 d NOEC 17.35 mg test item/L (Toxi-Coop, 2012)

Based on read-across approach, the calculated LOEC and NOEC values for lithium nitrate were 40 and 28 mg/L, respectively.

Supporting data

As laid down in Council Directive 98/83/EC a limit concentration of 50 mg/L nitrate ions (equivalent to 55.6 mg LiNO3/L) in drinking water was agreed. Also Directive 2000/60/EC and Council Directive 91/676/EEC refer to the limit value laid down in the Drinking Water Directive.

Further, it is published that the toxicity limit for fish regarding nitrate ions is around 100 -300 mg/L (equivalent to 111.2-333.6 mg LiNO3/L). (Haider, 1986)