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Ecotoxicological information

Long-term toxicity to fish

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Administrative data

fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
The Chronic Effects of Dietary Lead in Freshwater Juvenile Rainbow Trout (Oncorhynchus mykiss) Fed Elevated Calcium Diets
Alves LC & Wood CM
Bibliographic source:
Aquatic Toxicology 78: 217-232

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The study examined the impact of elevated Ca2+ on the responses to chronic dietary Pb exposure in juvenile rainbow trout. Only the data for those samples without the addition of lead are reported.
Trout were fed reference (0.3 µg Pb/g, ~20 mg Ca2+/g) diets in the presence of background (~20 mg Ca2+/g) or (~60 mg Ca2+/g) of added Ca2+ (as CaCO3) for 42 days.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium carbonate
EC Number:
EC Name:
Calcium carbonate
Cas Number:
Molecular formula:
calcium carbonate
Test material form:
solid: bulk

Test solutions

Details on test solutions:
Six replicated nominal dietary Pb and/or Ca2+ treatments:
(A) 0 μg Pb/g + 20 mg Ca2+ /g
(B) 0 μg Pb/g + 60 mg Ca2+ /g

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- Common name: Rainbow trout
- Source: Humber Springs Trout Hatchery (Orangeville, Ont)
- Age at study initiation (mean and range, SD): 20-23 g
- Weight at study initiation (mean and range, SD): mean weight of 25.8 ± 0.5 g
- Feeding during test: Yes
- Food type: Commercial salmon fry pellets
- Amount: 1.5% body mass/day
- Frequency: Once daily

- Acclimation period: 3 weeks

Study design

Test type:
Water media type:
Total exposure duration:
42 d

Test conditions

140 mg/L as CaCO3
Test temperature:
11-13 °C
Nominal and measured concentrations:
Nominal: 20 and 60 mg Ca2+/g dry weight
Measured: 19.2 and 61.0 mg Ca2+/g dry weight
Details on test conditions:
- Test vessel: 200 L polypropylene flow-through aerated tanks that were divided into 2x100 L sections
- Renewal rate of test solution (frequency/flow rate): 1 L/min of dechlorinated Hamilton water, pumped only once through the tanks.
- No. of organisms per vessel: 25 fish per section
- No. of vessels per control (replicates): 2 (Tanks 1 and 5)

- Source/preparation of dilution water: Dechlorinated Hamilton water

- Photoperiod: 12 h light and 12 h dark

EFFECT PARAMETERS MEASURED: Fish from each tank were bulk-weighed on days 0, 8, 15, 22, 29 and 36. Specific growth rates (SGR) expressed as a % per day were determined using linear regression of the natural logarithm of mean bulk weight versus time data. The food conversion efficiency (FCE, %) was also calculated.
On day 0, two randomly selected fish per tank were sacrificed. On days 14, 28 and 42, four randomly selected fish per tank section were also sacrificed. Blood samples were taken immediately. Vertebral bone, brain, gill baskets, anterior, mid and posterior intestine, anterior and posterior kidney, liver, muscle, spleen, stomach and remaining carcass were dissected out.
Blood samples were also taken on days 0, 7, 21 and 35.

Results and discussion

Details on results:
Growth and survival: Enhanced Ca2+ diets (60 mg Ca2+/g) had no effects on survival, despite a tendency for SGR to be lower in fish fed these diets. The mean fish weight (g) remained constant across all groups during the course of the experiment. FCE was significantly lower in the 0 µg Pb/g + 60 mg Ca2+/g compared to the 0 µg Pb/g + 20 mg Ca2+/g treatments.

Pb accumulation in tissues: Pb concentrations in the control treatment of all tissues were low (<0.05 µg/g tissue wet weight) but were significantly elevated on days 14 and 28 and occasionally on day 42, when compared to day 0.

Ion regulation and Na+, K+-ATPase activity: There were no significant differences observed in plasma Cl- and K+ in all treatments when compared to the controls over the duration of the experiment.

Applicant's summary and conclusion

Enhanced Ca2+ diets (60 mg Ca2+) had no effects on survival. Mean fish weights remained constant across all treatments.

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