Calcium nitrite

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-16 January 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Good quality study, conducted to GLP, with a slight deviation with regard to the timing of a single observation point. The test material was a 34% solution of hydrated calcium nitrite.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

Observations were made after 5 hours, and not after 6 hours, as stated in the protocol. This deviation was for practical reasons and did not affect the outcome of the study.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

Observations were made after 5 hours, and not after 6 hours, as stated in the protocol. This deviation was for practical reasons and did not affect the outcome of the study.

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in deionised water to give an initial 2 L stock solution (nominally at 1000 mg/L). This stock solution was added directly to the test vessel and made up to 20 L to give the desired exposure level.
- Eluate: not applicable
- Differential loading: not applicable
- Controls: untreated tap water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none reported

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Parkwood Trout Farm, Norfolk, UK
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): mean standard length was 4.9 cm, SD = 0.5 cm.
- Weight at study initiation (mean and range, SD): mean weight was 1.54 g, SD = 0.46 g.
- Method of breeding: not applicable
- Maintenance of the brood fish: not applicable

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): very similar
- Type and amount of food during acclimation: commercial fry pellet food
- Feeding frequency during acclimation: daily (except 24 hr period directly before exposure).
- Health during acclimation (any mortality observed): healthy (no mortality)


FEEDING DURING TEST
Fish were not fed during the 96 hour exposure period.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

As recommended in applicable guideline

Post exposure observation period:

Not applicable

Hardness:

130 mg/L

Test temperature:

14 +/- 1 deg C

pH:

mean 7.7

Dissolved oxygen:

mean 9.8 mg(O2)/L

Salinity:

Freshwater

Conductivity:

400 µS/cm

Nominal and measured concentrations:

Preliminary study: 0.1, 1, 10 or 100 mg/L (nominal)
Main study: 100 mg/L (nominal limit test)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria (25 x 46 x 25 cm).
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: each vessel contained 20 L of medium to a depth of 19 cm. This provided an initial loading of 0.77 g bodyweight/L (static volume).
- Aeration: "Supplementary aeration was provided via narrow glass tubes"
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): Daily batchwise renewal of test media.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap drinking water filtered, dechlorinated and softened by passage through a water purification system. Test substance dissolved in deionised water to give an initial 2 litre stock solution nominally at 1000 mg/L. This stock solution was added directly to the test vessel and made up to 20 L to give the desired exposure level.
Analysis of drinking water supply to Huntingdon Research Centre (6th November 1995):
- Total organic carbon: 1.4 mg/L
- Particulate matter: no data
- Metals: "not significant"
- Pesticides: Triazine herbicides (simazine, atrazine and propazine) all <0.03 ug/L; organophosphorus pesticides (including chlorpyrifos, diazinon, dichlorvos) all <0.02 ug/L; organochlorine pesticides (including aldrin, heptachlor, DDE and DDT) all <0.02 ug/L;
- Chlorine: 0.01 mg/L
- Alkalinity: 73 mg/L
- Ca/mg ratio: no data
- Culture medium different from test medium: no data
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: 16 hr light / 8 hr dark
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, measured by: (a) absence of respiratory movement and (b) absence of response to physical stimulation of the caudal peduncle. Subjective measurements on the type and incidence of sub-lethal effects, as compared to controls, were also made. Observations (lethal and sub-lethal) were made at 3, 5*, 24, 48, 72 and 96 hours. [The 5 hour exposure observation was a minor deviation from the protocol which indicated a 6 hour observation time point.]

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: limit concentration
- Range finding study
- Test concentrations: 0.1, 1, 10 or 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Effect level is equivalent to >34 mg/kg bw as calcium nitrite

Details on results:

In the main study, no mortalities were observed in rainbow trout exposed to water containing nominal test material concentrations of 100 mg/L for 96 hours. Sub-lethal effects were observed at this concentration, including 2 fish swimming at an angle, a fish lying on the bottom of the aquaria (at 48, 72 and 96 hours), 5 fish with darker pigmentation (at 24, 48 and 72 hours, reducing to 3 fish at 96 hours) and excess mucus production on the dorsal fin in all fish from 72 hours onwards.

In the preliminary study, no sublethal effects were seen at the lower concentration of 10 mg/L, except for one fish lying on the bottom of the aquaria at 48 hours. A concentration of 10 mg/L is therefore considered the study NOEC.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not applicable

Validity criteria fulfilled:

yes

Conclusions:

In a guideline study, to GLP, no mortality was apparent in freshwater rainbow trout exposed to water containing calcium nitrite (hydrate) at nominal limit concentration of 100 mg/L for 96 hours, although sub-lethal effects were observed.

Executive summary:

In a GLP study conducted to OECD Test Guideline 203 and EC Method C1, calcium nitrite (hydrate) was studied for acute toxicity to freshwater rainbow trout (Oncorhynchus mykiss), under semi-static conditions. The test substance (DCI), available as a 34% aqueous solution of calcium nitrite, was added to purified water and the fish exposed for 96 hours. In the preliminary study, test article concentrations of 0.1, 1, 10 or 100 mg/L (nominal) were used. No deaths were seen, so a main (limit) study was conducted using 10 fish/group exposed to 0 or 100 mg/L (nominal) for 96 hours.

In the main study, there was no mortality in rainbow trout exposed to water containing nominal test material concentrations of 100 mg/L for 96 hours. Sub-lethal effects were observed at this concentration, including two fish swimming at an angle, a fish lying on the bottom of the aquaria (at 48, 72 and 96 hours), 5 fish with darker pigmentation (at 24, 48 and 72 hours, reducing to 3 fish at 96 hours) and excess mucus production on the dorsal fin in all test-item exposed fish from 72 hours onwards. In the preliminary study, no sublethal effects were seen at the lower concentration of 10 mg/L, except for one fish lying on the bottom of the aquaria at 48 hours. Environmental parameters (including pH, temperature, and dissolved oxygen) remained within acceptable limits.

Based on these results, and in the absence of any mortality, the 96-hr LC50 for the test material was concluded to be >100 mg/L (the highest concentration tested). However, sub-lethal effects were seen at this concentration, but not at the lower concentration of 10 mg/L used in the preliminary test, so 100 mg/L can be seen as the test material study LOAEC.

Description of key information

In a GLP study conducted according to OECD Test Guideline 203, no mortality was apparent in freshwater rainbow trout exposed to water containing calcium nitrite (hydrate) at a nominal limit concentration of 100 mg/L (equivalent to 34 mg/L as calcium nitrite) for 96 hours, although sub-lethal effects were observed (Kelly, 1996a).

Additional information

In a GLP study conducted to OECD Test Guideline 203 and EC Method C1, calcium nitrite (hydrate) was studied for acute toxicity to freshwater rainbow trout (Oncorhynchus mykiss), under semi-static conditions. The test substance (DCI), available as a 34% aqueous solution of calcium nitrite, was added to purified water and the fish exposed for 96 hours. In the preliminary study, test article concentrations of 0.1, 1, 10 or 100 mg/L (nominal) were used. No deaths were seen, so a main (limit) study was conducted using 10 fish/group exposed to 0 or 100 mg/L (nominal) for 96 hours. In the main study, there was no mortality in rainbow trout exposed to water containing nominal test material concentrations of 100 mg/L for 96 hours. Sub-lethal effects were observed at this concentration, including two fish swimming at an angle, a fish lying on the bottom of the aquaria (at 48, 72 and 96 hours), 5 fish with darker pigmentation (at 24, 48 and 72 hours, reducing to 3 fish at 96 hours) and excess mucus production on the dorsal fin in all test-item exposed fish from 72 hours onwards. In the preliminary study, no sublethal effects were seen at the lower concentration of 10 mg/L, except for one fish lying on the bottom of the aquaria at 48 hours. Environmental parameters (including pH, temperature, and dissolved oxygen) remained within acceptable limits. Based on these results, and in the absence of any mortality, the 96-hr LC50 for the test material was concluded to be >100 mg/L (the highest concentration tested) [equivalent to >34 mg/L as calcium nitrite]. However, sub-lethal effects were seen at this concentration, but not at the lower concentration of 10 mg/L used in the preliminary test, so 100 mg/L can be seen as the test material study LOAEC (Kelly, 1996a).

 

The acute toxicity of sodium nitrite to fish varies widely between the species tested; 96-hr LC50 values ranging from 0.54 mg/L (for Oncorhynchus mykiss [rainbow trout]) to 1010.4 mg/L (for Anguilla japonica [Japanese eel] have been reported in the literature. This difference has been attributed to the ability of certain species, such as eels, bass and sunfish to prevent nitrite from crossing the gill membrane and entering the blood, whilst other species such as rainbow trout concentrate nitrite in their blood. Reported acute toxicity values also vary widely between studies on particular species of fish and the quality of the water used in the test (e.g. pH, calcium ion concentration and in particular chloride ion concentration) is considered able to influence the study results (OECD, 2005).