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Long-term toxicity to fish

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Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The study was not carried out according to GLP and the concentrations were measured as nitrate-nitrogen with a HACH-Lange reagens kit, which is a method that is not very specific. Furthermore the specific nominal and measured test concentrations were not reported. However, it is well described in a peer-reviewed journal. Therefore it is considered reliable with restrictions.
Qualifier:
according to guideline
Guideline:
other: American Society for Testing Materials. 1997. Standard guide for conducting early life-stage toxicity tests with fishes. E 1241-92. In Annual Book of ASTM Standards, Vol 11.05. West Conshohocken, PA, pp 550-577.
Deviations:
not specified
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
Concentrations were determined 4 to 6 times weekly
Vehicle:
no
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM

- Common name: Fathead minnow
- Source: Fertilized fathead minnow eggs were collected in-house from fathead minnow breeding pairs.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Hardness:
210 - 230 mg/L (CaCO3)
Test temperature:
23.0 ± 0.10 °C
pH:
7.75 ± 0.055
Dissolved oxygen:
>6.0 mg/L
Nominal and measured concentrations:
Five concentrations and a control were tested in duplo. However, it is not reported what concentrations were tested.
Details on test conditions:
These toxicity tests estimated the test chemical concentration that reduced hatch rate, fry survival, and fish weight over 32 d. Fertilized fathead minnow eggs were collected in-house from fathead minnow breeding pairs. When enough eggs were spawned on the same day, the eggs were consolidated into a beaker of control water by gently rolling them off the spawning substrate. Eggs were distributed in groups of five using a stratified-random procedure into egg cups suspended in the test aquaria (30x15x19cm, fill volume 6.75L, flow-through, renewal of test solution 24-25 times per day, peristatltic diluter, replacement of aquaria after 15d) with toxicant concentrations at the desired levels. Egg cups were constructed of glass jars that were 5 cm in depth and 5 cm in diameter. A stainless steel screen formed the bottom. Cups contained between 25 and 30 eggs each with two cups per aquarium giving four per treatment. The number of eggs was the same among treatments but varied among tests as spawning success of breeding pairs varied. As water levels in the aquarium rose and fell with each dosing apparatus cycle, water moved past the eggs, ensuring well oxygenated water and fresh toxicant. A 15:9 h light: dark photoperiod was provided. At 24-h intervals during the first 6 d, each egg cup was removed and placed in a Petri dish containing the test water, and the eggs were inspected under a magnifying glass. Dead eggs and those with fungus were recorded and removed. Hatched fry were removed from egg cups and placed in their appropriate test aquaria. All fry hatched within 6 d. Hatch success wasrecorded over the first 6 d as the percentage of successful hatch [hatch success = (total number of hatched fry/initial number of eggs) X 100]. Once hatched, fry were fed to satiation with freshly hatched brine shrimp ( <48 h old) augmented with finely ground flake food two to three times daily. Fry were counted on days 11, 18, 21, and 32, while transferring them with a large syringe from test aquaria into a temporary holding aquarium containing test water. Dead and missing fry, assumed to be dead, were recorded. Food was withheld 24 h prior to test conclusion. At the conclusion of the test, fish were given a lethal exposure to MS-222 and preserved in a 70% alcohol solution for approximately 2 weeks before weighing. Preserved individual fry weights were recorded to the nearest 0.001 g. Overall treatment effects on embryo and fry percentage survival (with arcsine transformation) and mean fry weight were determined using one-way ANOVA. Each treatment was compared to the control using Dunnett's test. The NOEC and LOEC were reported as in the growth tests.
Reference substance (positive control):
not specified
Key result
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
157 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
other: NO3-N
Basis for effect:
growth rate
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
294 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
other: mg NO3-N
Basis for effect:
growth rate
Details on results:
The NOEC and LOEC were based on a significant reduction (p = 0.027) in weight of fish at the end of the test ( 11 mg in the middle treatment vs 16 mg for the controls). Although fry survival was significantly reduced (p = 0.007) from the control (23% survived vs 78% for the control), that reduction occurred at a higher nitrate concentration than for final weight. Nitrate concentrations had no effect on embryo survival.
Validity criteria fulfilled:
not specified
Conclusions:
NOEC 157 mg/L (growth rate)
LOEC 294 mg/L (growth rate)
Endpoint:
fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The study was not carried out according to GLP and the concentrations were measured as nitrate-nitrogen with a HACH-Lange reagens kit, which is a method that is not very specific. Furthermore the specific nominal and measured test concentrations were not reported. To reduce reproductive behaviour, the photoperiod of the growth test was 9:15 (light:dark), which deviates from the usual 16:8 cycle. However, it is well described in a peer-reviewed journal. Therefore it is considered reliable with restrictions.
Principles of method if other than guideline:
Growth tests estimated the test chemical concentration that affected fish growth over 30 d. Specific growth rate (SGR) was measured as percentage growth per day (SGR = [log e (final measure - initial measure)]/[number of days elapsed between measurements]). Twelve fish, acclimated to test temperatures, were distributed to each test aquarium in a stratified-random manner. Fish were then weighed to the nearest 0.01 g, and total length was measured to the nearest 0.5 mm. During this process, fish received one of 12 unique fin clips to allow identification of individual fish. Fin clips used were dorsal, anal, left or right pectoral, left or right pelvic, upper or lower caudal, or combinations of caudal with pectoral or caudal with pelvic. Preliminary experiments showed that fin clips did not significantly reduce the growth rate of test fish over a 30-d period. Before fish were weighed, food was withheld for 24 h. During the measuring and fin clipping process, fish were anesthetized with 100 mg/L tricaine methanesulfonate (MS-222). Once measured, weighed, and clipped, fish were allowed 24 h to acclimate to test aquaria prior to chemical exposure. Test fish received a refresh of the fin clip and were reweighed at 15 d after withholding food for 24 h. The toxicity test concluded with a final weight and length measurement at 30 d after withholding food for 24 h. During the testing period, fish were fed to satiation two or three times
per day using a combination of frozen adult brine shrimp and flake food. Uneaten food was siphoned from test aquaria at least once daily. An overall effect of the treatment on percentage survival (with arcsine transformation) and SGR was determined using one-way ANOVA. Each treatment was compared to the control using Dunnett's test. Three endpoints were described: the no-observed-effect concentration (NOEC), which was the highest concentration not significantly different from the control (p < 0.05); the lowest-observed-effect concentration (LOEC), which was the lowest concentration that was significantly different from the control (p < 0.05).
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
Concentrations were determined 4 to 6 times weekly
Vehicle:
no
Test organisms (species):
other: juvenile Topeka shiner and with juvenile Fathead minnow
Details on test organisms:
TEST ORGANISM

The test was carried out with juvenile Topeka shiner and with juvenile Fathead minnow

- Common name: Topeka shiner
- Source: Juvenile (~7 months) Topeka shiners were obtrained annually over a 3-year period from Lost Valley Fish Hatchery in Missouri, USA. They were held at appr. 12 °C in 1,500 L flow-through aerated holding tanks until the acclimation period. During the holding period, fish were fed frozen brown shrimp (Artemia salina) augmented with flake food once or twice daily.
- Age at study initiation: 10 months (juveniles)
- Length at study initiation: Not reported
- Weight at study initiation: 0.79 ± 0.02 g

- Common name: Fathead minnow
- Source: Fathead minnows were bred in-house and originated from a mixed stock from Kurtz Fish Hatchery, Elverson, Pennsylvania, USA, and the U.S. EPA, Duluth, Minnesota, USA. Fathead minnows were fed the same diet as the Topeka shiners and were held at approximately 25°C.
- Age at study initiation: 7 months (juveniles)
- Length at study initiation: Not reported
- Weight at study initiation: 0.73 ± 0.012 g

ACCLIMATION
- Acclimation period: Once measured, weighed, and clipped, fish were allowed 24 h to acclimate to test aquaria prior to chemical exposure.
- Acclimation conditions: same as test
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 d
Hardness:
210 - 230 mg/L (CaCO3)
Test temperature:
Topeka shiner: 23.4 ± 0.04 °C
Fathead minnow: 23.1 ± 0.07 °C
pH:
8.25 ± 0.012
Dissolved oxygen:
Topeka shiner: >6.0 mg/L
Fathead minnow: 5.5 mg/L
Nominal and measured concentrations:
Five concentrations and a control were tested in duplo. However, it is not reported what concentrations were tested.
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria with a stainless steel screen at one end maintaining water depth at 22 cm
- Material: Not reported
- Dimensions: 50 x 25 x 30cm
- Fill volume: 27.5 L
- Type of flow-through (e.g. peristaltic or proportional diluter): Peristaltic diluter
- Renewal rate of test solution (frequency/flow rate): 6 - 7 times per day
- Replacement of aquaria: After 15 days, test aquaria were replaced with clean aquaria
- No. of organisms per vessel: 12
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates): 2
- Biomass loading rate: Topeka shiner: 0.34 g/L, Fathead minnow: 0.32 g/L (Calculated by reviewer based on average initial weight and fill volume)

TEST MEDIUM / WATER PARAMETERS
- Source: A deep well (not further specified), total alkalinity, chloride concentration and pH were recorded

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 9:15 (light dark) to reduce reproductive behaviour
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED: Survival and specific growth rate (intervals not specified)

TEST CONCENTRATIONS
No information provided on test concentrations
Reference substance (positive control):
not specified
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
268 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
other: NO3-N
Basis for effect:
growth rate
Remarks on result:
other: Juvenile Topeka shiner
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
486 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
other: NO3-N
Basis for effect:
growth rate
Remarks on result:
other: Juvenile Topeka shiner
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
58 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
other: NO3-N
Basis for effect:
mortality
Remarks on result:
other: Juvenile Fathead minnow
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
121 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
other: NO3-N
Basis for effect:
mortality
Remarks on result:
other: Juvenile Fathead minnow
Details on results:
Juvenile Topeka shiner: The NOEC and LOEC were based on a significant reduction (p = 0.007) in specific growth rate at 30 d . None of the treatments had a significant effect on survival.
Juvenile Fathead minnows: The NOEC and LOEC were based on a significant reduction (p ≤ 0.001) in survival (38% survived in the third highest treatment vs 100% in controls) at 30 d The specific growth rate of fathead minnows at 121 mg/L N was significantly higher than that of fish in the controls (p = 0.028). A reduced density of fish in the aquarium may have caused the higher specific growth rate in that treatment.
Validity criteria fulfilled:
not specified
Conclusions:
Juvenile Topeka shiner: NOEC 268 mg/L, LOEC 486 mg/L
Juvenile Fathead minnow: NOEC 58 mg/L, LOEC 121 mg/L

Description of key information

In accordance with column 2 of REACH Annex IX, no long term toxicity testing is proposed (required in section 9.1.6) as the chemical safety assessment does not indicate a need to further investigate the effects on fish. All data available on magnesium nitrate itself and on the other nitrates show a very low toxicity. In addition, the substance does have a very high water solubility and its chemical properties do not indicate bioaccumulation. Therefore, the study is not considered necessary.

Key value for chemical safety assessment

Additional information

Although the endpoint is waived, there are reliable 30d growth rate and 32d embryo-larval tests available for the structural related substance sodium nitrate from Adelman (2009).

In the 30d growth rate test the NOEC for juvenile Topeka shiner was 268 mg/L (growth rate) and the NOEC for Fathead minnow was 58 mg/L (mortality).

In the 32d embryo-larval test, the NOEC to Fathead minnow was 157 mg/L based on growth rate (no effect on embryo survival).

These tests confirm the low toxicity of sodium nitrate.

The read-across rationale can be found in the document attached in the target record.