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Long-term toxicity to fish

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fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remarks'
The result was obtained from a collection of reliable data which has been subject to peer-review and in which the original data sources are traceable.The study was well documented and meets generally accepted scientifically sound principles, but was not conducted in compliance with GLP.
Principles of method if other than guideline:
Six 15L tanks fitted with egg-holding trays and downstream weirs so that gentle flow of water/test solution maintained around eggs. Fry could fall between glass rods supporting eggs, but couldn't leave tanks due to mesh screens. Opaque tanks and lids. Data provided on mortality at egg, larval and juvenile (fry) stages; length of time from hatching to full resorption of yolk; abnormalities in development.
GLP compliance:
not specified
The data are from a published source and it is not clear whether or not the study was conducted in compliance with GLP
Analytical monitoring:
Details on sampling:
- Concentrations: Measured Ammonia as N (mg¯¹): Total: 0.14,2.55, 4.52, 8.00, 15.50 and 25.77
Calculated Undissociated Ammonia as N (mg¯¹): 0.022, 0.039, 0.069, 0.128 and 0.219
- Sampling method: Not reported
- Sample storage conditions before analysis: Not reported
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Analytical grade ammonium chloride was delivered in aqueous stock solution to free-piston burettes whose action was controlled by concentric-siphon dosers (Shurben 1978). The dosers mixed the stock solution with dilution water and passed it to the test vessels.
Dilution water was a natural, hard, unchlorinated, borehole water.
Test A: eggs exposed to test material from within one day after fertilisation.
Test B: eggs exposed to test material 24 days after fertilisation when the eggs reached the eyed stage.

- Controls: One control vessel with dilution water but no test material.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not reported
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- Common name: Rainbow trout
- Strain: Not reported
- Source: Not reported

- Numbers of parental fish (i.e. of females used to provide required number of eggs): Not reported
- Method of collection of fertilised eggs: Not reported
- Subsequent handling of eggs: Not reported

- Start date: Not reported
- Type/source of feed: Proprietary fry food
- Amount given: Not reported
- Frequency of feeding: Not reported
Test type:
Water media type:
Limit test:
Total exposure duration:
73 d
Remarks on exposure duration:
Also Test B: 49 day exposure test for eggs exposed to test material 24 days after fertilisation
263 +/- 2.3 as CaCO3
Test temperature:
Range 11.5 to 17.0 °C
Control: 7.52 ± 0.05
Control pH values were never more than 0.07 pH units higher than the lowest value from the test vessels.
Dissolved oxygen:
Pre-hatching stages: ≥ 95% ASV
Larval and Post larval periods: 92 and 76% ASV
Nominal and measured concentrations:
Measured: Total concetration of Ammonia as N (mg¯¹):: 0.14,2.55, 4.52, 8.00, 15.50 and 25.77
Calculated Concentration of Undissociated Ammonia as N (mg¯¹): 0.022, 0.039, 0.069, 0.128 and 0.219
Details on test conditions:
- Emybro cups (if used, type/material, size, fill volume): Egg holding trays and downstream weirs so gentle flow of water or test solutions was maintained around the eggs.
- Test vessel: 15 litre capacity tanks
- Type (delete if not applicable): open / closed - lidded (lids partially reomved before the fry began to feed independently.
- Material, size, headspace, fill volume: Opaque with opaque lids. No other information.
- Aeration: Yes - no details
- Type of flow-through (e.g. peristaltic or proportional diluter): Stock solution delivered to free-piston burettes controlled by concentric-siphon dosers.
- Renewal rate of test solution (frequency/flow rate): Dosers mixed the stock solution with dilution water and passed it to test vessels
- No. of fertilized eggs/embryos per vessel: Test A: 591-737 eggs per vessel
Test B: 136-174 eggs per vessel.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: Not reported

- Source/preparation of dilution water: A natural, hard, unchlorinated borehole water
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: 243 +/- 3.2 mg L¯¹
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Salinity: Freshwater
- Culture medium different from test medium: Not reported
- Intervals of water quality measurement: Not reported

- Adjustment of pH: pH controlled using small additions of carbon dioxide to the aeration flow as necessary.
- Photoperiod: Not reported
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The following effect parameters were observed daily:
i) Mortality at egg, larval and juvenile (fry) stages
ii) The length of time from fertilisation to hatching
iii) The length of time from hatching to full resorption of yolk
iv) Abnormalities in development

- Begin of post-hatch period: Not reported
- No. of hatched eggs (alevins)/treatment released to the test chamber: Not reported
- Release of alevins from incubation cups to test chamber on day no.: Not reported

- Number of eggs used: Not reported
- Removal of eggs to check the embryonic development on day no.: Not reported
73 d
Dose descriptor:
Effect conc.:
0.022 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: undissosiated ammonial as N
Basis for effect:
73 d
Dose descriptor:
Effect conc.:
0.027 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: unionised ammonia as NH3
Basis for effect:
73 d
Dose descriptor:
Effect conc.:
0.014 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: unionised ammonia as NH3
Basis for effect:
Details on results:
The data for periods to eyeing, hatching or resorption of yolk and for dry-matter content varied little between treatments and no concentration-related sub-lethal responses could be identified
Reported statistics and error estimates:
Not reported

Table 1. Test Conditions and responses of early life satges of rainbow trout to ammonia - mean values, with SD in parentheses

 Concentrations of ammonia as N (mg ¯¹)     Mortality % individuals entering stage         
 Total Undissociated   Egg  Yolk-sac fry  Fry  Mortality (total %*)
 Test A (started within 24 h of fertilisation)               
 0.14 (0.17)  -  8.8  1.6  2.6  12.6
 2.55 (0.41)  0.022 (0.004)  65.2  12.6  4.9  71.1
 4.52 (0.43)  0.039 (0.009)  78.5  33.6  2.7  86.1
 8.00 (0.49)  0.069 (0.013)  87.3  44.1  0  92.9
 15.50 (0.57)  0.128 (0.012)  72.1  26.8  4.7  80.6
 25.77 (1.65)  0.219 (0.040)  97.9  61.5  20.0  99.4
 Test B (started 24 days after fertilisation)            
0.19 (0.20)  -  2.2  4.0  3.6  9.5
 2.59 (0.39)  0.023 (0.004)  3.3  9.6  11.6  22.7
 4.74 (0.19)  0.041 (0.10)  3.2  5.7  2.5  11.0
 8.15 (0.49)  0.072 (0.015)  3.4  3.8  3.5  10.3
 15.72 (0.53)  0.130 (0.012)  3.4  11.2  8.3  21.3
 25.92 (2.28)  0.226 (0.046)  3.3  14.0  27.5  39.7

* The "total mortality" was calculated separately to take account of the eggs and fry removed from each vessel for the measurement of dry matter content.

Validity criteria fulfilled:
not specified
A 73 day LOEC of 0.022 mg l¯¹ as N (0.027 as NH3) has been determined for the effects of undissociated ammonia on mortality of Oncorhynchus mykiss eggs, larvae and fry. A factor of 2 can be applied to the LOEC to estimate a NOEC. This gives a NOEC value of 0.014 mg NH3/L.

Description of key information

Long-term toxicity to fish: 73-day LOEC 0.027 mg NH3/l and NOEC 0.014 mg NH3/l as calculated from the LOEC with a factor of 2, (measured) (no test guideline followed), for ammonia.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.014 mg/L

Additional information

Reliable long-term data are available sourced from the Environment Agency Proposed EQS for Water Framework Directive Annex VIII Substances: Ammonia (unionised) 2007.

The lowest credible concentration of unionised ammonia at which long-term effects were found is 0.022 mg/l of nitrogen present as NH3 (0.027 mg NH3/l) when a cumulative mortality of 71 per cent was observed for eggs, larvae and fry of rainbow trout (Oncorhynchus mykiss) over 73 days exposure (Solbe et al, 1989). No NOEC value was derived in the study since effects were observed at the lowest exposure concentration. However, data on the concentration–response curve for similar effects in other fish have been evaluated, and these indicate 2–3 times difference in exposure concentration for a 50 per cent reduction in survival (from the controls) in such an early life stage test (EA 2007). A factor of 2 is applied to the LOEC result to estimate a NOEC, giving a long-term toxicity to fish result of 0.014 mg unionised NH3/l.