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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 24, 2000 - November 30, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Purity of test substance unknown.
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material: no data
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal concentrations of 6.25, 12.5, 25.0, 50.0, and 100 mg/L
- Sampling method: Water samples were collected at 0 (test initiation) and 96 hours (test termination) of the definitive toxicity test.
Control and test substance-fortified samples were also analyzed at each sample point.
At each sampling point, 6-mL samples were collected from each replicate of the control or exposure treatments and composited in culture tubes.
Samples were collected subsurface from the approximate center of each exposure chamber. At termination, an additional 500-mL control
composite sample was collected by combining approximately 250 mL of each control replicate in a 1-L beaker for preparation of quality control
fortification samples (QC spikes). Two QC spikes were prepared at each sample point at concentrations ranging from 3.13 to 106 mg MAP/L.
The low and high QC spikes were prepared by diluting MAP with dilution water at test initiation and with the control composite sample
at test termination. Samples were diluted, if necessary, with either dilution water (at initiation) or control composite sample (at termination)
so that the expected analyte concentrations were within the range of the calibration curve (1.00 to 20.0 mg total phosphate/L).
Vehicle:
no
Details on test solutions:
no data
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: obtained as fertilized eggs from Troutlodge, Inc., Sumner, WA, USA
- Age at study initiation (mean and range, SD): approximately five months
- Length at study initiation (length definition, mean, range and SD): 45 to 60 mm total length (mean and standard deviation = 53 ± 5.5 mm)
- Weight at study initiation (mean and range, SD): 0.662 to 1.768 g wet weight (mean and standard deviation = 1.226 ± 0.407 g)
- Method of breeding: Following hatch, fish were fed salmon starter and/or brine shrimp nauplii daily during the holding period.
Three days prior to test initiation, approximately 230 fish were removed from the holding tank and placed in acclimation.
- Feeding during test: fish were not fed during the test.


ACCLIMATION
- Acclimation period: 3 days
- Acclimation conditions (same as test or not):Acclimation water temperature ranged from 15.2 to 15.9°C.
- Type and amount of food: animals were not fed during acclimation
- Health during acclimation (any mortality observed): no mortality and no diseases observed.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No data.
Hardness:
160 mg/L as CaCO3 at initiation
Test temperature:
15 ± 1°C
pH:
7.10 - 7.80
Dissolved oxygen:
Equal to or greater than 8.54 mg/L (equal to or greater than 88% of saturation) at initiation of the definitive test.
Levels of some of the test solutions were < 60% of saturation on study days 2 and 3, but did not fall below 5.63 mg/L in any chamber.
From study day 3 through termination, all solutions were gently aerated.
Levels were equal to or greater than 7.42 mg/L (76% of saturation) at termination.
Salinity:
Conductivity and temperatures were determined (see 'Test temperature' and 'Details on test conditions')
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25.0, 50.0, and 100 mg/L
Measured concentrations: 5.72, 11.9, 23.5, 46.6, and 85.9 mg/L (mean of t=0 and 96 h; 86 to 95% of nominal concentration)
The concentration of total phosphate in the control samples were detected at less than MDL (0.389 mg total phosphate/L or 0.598 mg MAP/L)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 5-gallon glass jars containing 15 L of control or test solution and covered with clear plastic petri dish
covers. The glass jars had measurements of approximately 47 cm (height) by 26 cm (inside-diameter) with 29 cm solution depth.
- Aeration: From study day 3 through termination, all solutions were gently aerated.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: Instantaneous loading was calculated to be 0.41 g of fish tissue per liter of test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was a moderately hard laboratory freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis. These waters were blended to yield a total hardness of 130 to 160 mg/L as CaCO3.
The water was filtered prior to use.
- Alkalinity: 150 mg/L as CaCO3
- Conductivity: 304 uS/cm
- Total hardness: 160 mg/L as CaCO3
- Ammonia: Control samples, measured as total ammonia, were determined to be 0.062 and <0.012 mg/L on day 0 (test initiation)
and 0.28 and 0.30 mg/L on day 4 (test termination). Treatment samples, measured as total ammonia, increased proportionately with test
substance concentration and ranged from 1.2 to 18 mg/L on day 0 and from 0.66 to 18 mg/L on day 4.
Control samples, measured as un-ionized ammonia, were determined to be 0.0011 and <0.00023 mg/L on day 0 and both replicates
were 0.0036 mg/L on day 4. Treatment samples, measured as un-ionized ammonia, increased proportionately with test substance concentration and
ranged from 0.019 to 0.065 mg/L on day 0 and from 0.0069 to 0.16 mg/L on day 4.
- Intervals of water quality measurement: Temperature, dissolved oxygen, and pH were measured initially (prior to addition of fish) and daily
thereafter during the conduct of the test.In addition, total hardness, total alkalinity, and conductivity were measured at initiation of the definitive test.
All control and treatment solutions were analyzed for both total and un-ionized ammonia concentrations at initiation and termination of the test.


OTHER TEST CONDITIONS
- Photoperiod: Fluorescent lighting was maintained on a 16-hour daylight photoperiod with 30-minute simulated dawn and dusk periods.
- Light intensity: 439 - 473 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
mortality and sublethal responses at test initiation and once every 24 ± 1 hours thereafter for the remainder of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: nominal total product test concentrations of 0.0 (control), 0.1, 1.0, 10, and 100 mg/L. Five fish were tested per
treatment.
- Results used to determine the conditions for the definitive study: No mortality or sublethal effects were observed in the control or any treatment
level following 96-hours of exposure.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Basis for effect:
mortality (fish)
Remarks on result:
other: Nominal concentration confirmed by chemical analysis (>80% recovery)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Basis for effect:
other: mortality and sublethal effects
Remarks on result:
other: Nominal concentration confirmed by chemical analysis (>80% recovery)
Details on results:
No lethal or sublethal effects were observed at the highest concentration tested.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
Statistical analysis of the concentration versus effect data (mortality) was not performed due to the absence of mortality during the definitive test.
The slope of the 96-hour dose-response line (log concentration vs. probit) could not be calculated due to the absence of fish mortality.
Sublethal observations / clinical signs:

none

Validity criteria fulfilled:
yes
Conclusions:
Based on mean calculated total product test concentrations of Monoammonium Phosphate in test water, the nominal 96-hour LC50 value was determined to be >100 mg/L , the highest treatment concentration of the definitive test. The nominal 96-hour NOEC was 100 mg/L, based on the absence of any mortality and sublethal effects at this and all lower treatment concentrations. Nominal concentrations were confirmed by chemical analysis (>80% recovery). The slope of the 96-hour dose-response line could not be calculated due to the absence of mortality.

Description of key information

Based on a reliable study with ammonium dihydrogenorthophosphate the nominal LC50 for freshwater fish (rainbow trout) is > 100 mg/L. Nominal concentrations were confirmed by chemical analysis (>80% recovery). 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

In a reliable OECD 203 guideline study, rainbow trout were exposed for 96 hr under static conditions to 6.25, 12.5, 25.0, 50.0, and 100 mg/L ammonium dihydrogenorthophosphate. Measured concentrations were determined to be 5.72, 11.9, 23.5, 46.6 and 85.9 mg/L (mean of t=0 and 96 h; 86 to 95% of nominal concentration). Based on mean calculated total substance concentrations in test water, the nominal 96-hour LC50 value was determined to be >100 mg/L, the highest treatment concentration of the definitive test. The nominal 96-hour NOEC was ≥100 mg/L, based on the absence of any mortality and sublethal effects at this and all lower treatment concentrations. Nominal concentrations were confirmed by chemical analysis (>80% recovery). The slope of the 96-hour dose-response line could not be calculated due to the absence of mortality.