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Long-term toxicity to fish

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fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
supporting study
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
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:
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 (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
Test type:
Water media type:
Total exposure duration:
42 d
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.
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.
Enhanced Ca2+ diets (60 mg Ca2+) had no effects on survival. Mean fish weights remained constant across all treatments.
long-term toxicity to fish
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:

Description of key information

No reliable long-term studies on fish are available.

Key value for chemical safety assessment

Additional information

Both calcium and carbonate ions are extremely common in all natural surface waters and are therefore ubiquitous in the environment. Seawater contains approximately 400 ppm calcium and rivers generally contain 1-2 ppm calcium but in lime areas rivers may contain calcium concentrations as high as 100 ppm (Lenntech, 2010). One of the main reasons for the abundance of calcium in water is due to its natural occurrence in the Earth’s crust. These calcium-rich rocks undergo physical and/ or chemical weathering in the environment. The calcium sediments are then carried by water from the mountains to oceans or lakes as well as to the land portion of the Biosphere and thereby become part of the calcium and carbon cycles. This natural abundance of calcium and carbonate ions in the environment means that aquatic organisms including fish are constantly exposed to calcium carbonate without suffering from any adverse or detrimental effects.

Furthermore, both calcium and carbonate are essential constituents of living organisms, including fish.

Calcium carbonate is also directly applied to lakes to mitigate the effects of surface water acidification and to ensure the survival of aquatic species (Driscoll et al 1987). This use therefore implies that calcium carbonate is not toxic to fish following long term exposure. A 6 year study by Popp et al (1996) demonstrated that the addition of calcium carbonate to a slightly acidic lake did not adversely affect the integrity of the fish community and in fact may have increased the abundance and biomass of the forage fish community and indirectly increased the survival, abundance and growth of brook trout.

An acute toxicity study to Oncorhynchus mykiss (rainbow trout) was performed according to OECD 203 with a saturated solution (100% v/v) of uncoated nano calcium carbonate (Priestly, 2010). No mortalities or sub-lethal effects were observed in any of the fish exposed. As a result, calcium carbonate is considered not acutely toxic to fish and long term testing is considered to be unnecessary.

Two literature references, used as supporting data, indicate that exposure to calcium carbonate has no effects on the long-term survival of fish (Morgan et al, 2005 and Alves and Wood, 2006).