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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:
5 October 2007 to 9 October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study followed existing guideline and GLP requirements
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1085 (Fish Acute Toxicity Test mitigated by humic acid)
Deviations:
yes
Remarks:
fish weight range lower than protocol, but biomass to solution ratio in range; fish were not fed 24 hours before test rather than 48 hours before
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accommodating fraction of a 100 mg active ingredient primary stock solution was prepared by placing 3.2824 grams SD-10 (3.1905 grams as active ingredient) in 32 liters of dilution water. The stock solution was mixed using a magnetic stir plate and Teflon-coated stir bar for approximately 3 hours. The initial pH of the stock solution was 8.5 and was adjusted to 7.0 by the addition of 5.0 N hydrochloric acid (total volume = 0.80 ml). An oily film was observed on the surface of the resultant stock solution. The soluble portion of this stock solution was removed by siphon and used to prepare the test solutions.
Each exposure solution was prepared by diluting the appropriate volume of 100% WAF stock solution to a final volume of 15 L with dilution water, and mixing with a glass rod for approximately 30 seconds.
- Controls: Control aquaria contained dilution water only in the 0 mg humic acid/L test; the tests with humic acid used a dilution water control and a dilution water plus humic acid at 10 or 20 mg humic acid/L.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Stock solution was water accommodating fraction of a 100 mg ai/L primary stock solution. Nominal concentrations for all three tests (0 mg humic acid/L, 10 mg humic acid/L and 20 mg humic acid/L) were expressed in percent of the Water Accommodating Fraction and were 100, 50, 25, 13 and 6.3% WAF and control. The US EPA interpreted these concentrations to correspond to 3.2 mg ai/L (water solubility), 1.6 mg ai/L, 0.80 mg/L, 0.42 mg/L, 0.20 mg/L, and 0 mg ai/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Oily film on surface of stock solution. Soluble portion of stock solution used to prepare dilutions was clear and colorless with no visible undissolved material.

Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: Pimephales promelas, SSL Lot No. 07A134
- Source: Springborn Smithers Laboratories culture facility
- Length at study initiation (length definition, mean, range and SD): N = 30 fish from test population had mean weight of 0.15 g (range 0.10 to 0.21 g)
- Weight at study initiation (mean and range, SD): N = 30) from test population had mean total length of 25 mm (range 24 to 26)
- Feeding during test: no


ACCLIMATION
- Acclimation period: Minimum of 9 days prior to definitive test
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: live brine shrimp prior to testing, not fed 24 hours before test or during exposure
- Feeding frequency: at least once daily prior to testing.
- Health during acclimation (any mortality observed): No mortality in test fish population during any 7-day period prior to testing

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Dilution water hardness was characterized for total hardness as CaCO3 of 38 to 46 mg/L.
Test temperature:
22 +/-1 degree C See tables in materials and methods
pH:
See tables in materials and methods
Dissolved oxygen:
See tables in materials and methods
Nominal and measured concentrations:
0, 6.3, 13, 25, 50 and 100 % Water Accommodating Fraction nominal concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria
- Material, size, headspace, fill volume: glass and silicone adhesive constructed aquaria, 39 x 20 x 25 cm, 15 liters fill volume
- Aeration: only if dissolved oxygen levels dropped below acceptable values
- No. of organisms per vessel: 10 fish per aquarium
- No. of vessels per concentration (replicates): one replicate for each of the 5 concentrations and controls; three test conditions (0 mg/l, 10 mg/l and 20 mg/l humic acid)
- No. of vessels per control (replicates): one replicate per control; humic acid tests had one dilution water control and one dilution acid plus humic acid control
- Biomass loading rate: 0.10 g biomass per liter test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well water from Springborn Smithers and Town of Wareham, pH 6.3 to 7.0
- Total organic carbon: TOC of dulution water source ranged from 0.32 to 0.49 mg/L from April to September, 2007. (Source water analyzed monthly)
- Metals: None detected in toxic amounts as analyzed in agreement with ASTM (2002) standard practice
- Pesticides: None detected in toxic amounts as analyzed in agreement with ASTM (2002) standard practice
- Alkalinity: 21 to 22 mg/L as CaCo3
- Conductivity: 200 umhos/cm
- Culture medium different from test medium: No, except for humic acid additions in two test conditions
- Intervals of water quality measurement: Dilution water source tested periodically for above water parameters

OTHER TEST CONDITIONS
- Adjustment of pH: initial pH of stock solutions adjusted to pH of 7.0 with 5.0 N hydrochloric acid
- Photoperiod: 16 hours light/ 8 hours dark
- Light intensity: 30 to 100 footcandles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality at 24, 48, 72 and 96 hours; physical and behaviorial abnormalities (lethargy, loss of equilibrium. Calculations of LC50 values and NOEC values for those intervals.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Each test concentration was to be at least 50% of the next higher concentration (dilution factor not to exceed 2.0).
- Range finding study: Yes; 5 fish per aquaria; 0 mg/l, 10 mg/l and 20 mg/L humic acid tests
- Test concentrations: 0.010, 0.10, 1.0, 10 and 100% water accommodating fraction of 100 mg ai test article/ Liter
- Results used to determine the conditions for the definitive study: No mortality nor observational adverse effects seen in any treatment or control
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
35 other: % Water Accommodating fraction
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
0 mg/l added humic acid
Basis for effect:
mortality
Remarks on result:
other: 95% confidence intervals of 25 and 50% WAF of a 100 mg ai/L test solution
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
25 other: % Water Accommodating Fraction
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
0 mg/L added humic acid
Basis for effect:
behaviour
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 other: % Water Accommodating Fraction
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
10 mg/L added humic acid
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
50 other: % Water accommodating fraction
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
10 mg/L added humic acid
Basis for effect:
behaviour
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 other: % Water Accommodating Fraction
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
20 mg/l added humic acid
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 other: % Water Accommodating Fraction
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
20 mg/L added humic acid
Basis for effect:
behaviour
Reported statistics and error estimates:
Nominal concentrations tested and the corresponding mortality data from the definitive test were used to estimate the median lethal concentrations (
LC50) and 95% confidence intervals. The LC50 was defined as the concentration of the test substance in dilution water which caused mortality of 50% of the test organism population at the stated time interval. If at least one test concentration caused mortality equal or greater than 50%, then a computer program (Stephan, 1982) was used to calculate the LC50 and 95% CL values. Three statistical methods were available in the program: moving average angle analysis, probit analysis and binomial probability. Moving average angle and probit analysis yield sound results only if at least 2 concentrations cause mortality between 0 and 100%. Selection criteria for reported LC50 values included establishment of dose-response relationship, number of concentrations causing partial effects, and span of responses bracketing the EC50. If two or more methods produced acceptable results, the method with smallest 95% CL was chosen. NOEC for the 96 hour exposure was also determined.

Mortality and observations

Nominal

Conc. (%WAF)

Humic acid (mg/L)

0 hour

2 hour

3 hour

6 hour

24 hour

48 hour

72 hour

96 hour

Cumulative Mortality (%) a

Control

0

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

6.3

0

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

13

0

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

25

0

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

50

0

0 (0)

0 (0)

0 (0)

0 (0)

90 (9)b

100 (10)

100 (10)

100 (10)

100

0

100 (10)

100 (10)

100 (10)

100 (10)

100 (10)

100 (10)

100 (10)

100 (10)

Control

0

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

Humic control

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

6.3

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

13

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

25

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

50

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

100

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)b

0 (0)b

Control

0

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

Humic control

20

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

6.3

20

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

13

20

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

25

20

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

50

20

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

100

20

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)c

0 (0)c

0 (0)c

0 (0)c

a Actual number of mortalities is presented I parenthesis

b All surviving fish were observed to be lethargic

c Humic acid was observed to have precipitated out of solution

LC50 values, with 95% confidence intervals, and No Observable Effect Concentrations during 96 hour exposures of fathead minnows to SD-10

95% Confidence Intervals

LC50 (%WAF)

Lower (% WAF)

Upper (% WAF)

0 mg humic acid/L

24 hour b

38

25

50

48 hour b

35

25

50

72 hour b

35

25

50

96 hour b

35

25

50

NOEC through 96 hours = 25% WAF of a 100 mg a.i./L solution

10 mg humic acid/L

24 hour

> 100

NA c

NA

48 hour

> 100

NA

NA

72 hour

> 100

NA

NA

96 hour

> 100

NA

NA

NOEC through 96 hours = 50 % WAF of a 100 mg a.i./L solution

20 mg humic acid/L

24 hour

> 100

NA

NA

48 hour

> 100

NA

NA

72 hour

> 100

NA

NA

96 hour

> 100

NA

NA

NOEC through 96 hours = 100 % WAF of a 100 mg a.i./L solution

a  Nominal concentrations are presented as percent of a 100 mg a.i./L water accommodated fraction (WAF)

b  LC50 value estimated by binomial probability

c  NA = Not applicable. LC50 value was empirically estimated to be greater than the highest nominal concentration tested; thus, confidence intervals could not be calculated.
Validity criteria fulfilled:
yes
Conclusions:
Test item in pure dilution water produced lethality and lethargy in fathead minnows exposed under static conditions for up to 96 hours. 96 hour LC50 was 35% Water Accommodating Fraction of a 100 mg active ingredient/L test solution (95% Confidence Interval 25 to 50% WAF). 96 hour NOEC for that test condition was 25% WAF of a 100 mg ai/L test solution. Mortality and behavioral effects of the same test concentrations were mitigated by adding humic acid to the dilution waters. No mortality was seen at 100% WAF of a 100 mg ai/L test solution with the addition of 10 or 20 mg/L humic acid. At 10 mg/L, lethargy was seen in fish exposed to 100 mg ai/L, but not lower concentrations. NOEC for that test condition was 50% WAF of a 100 mg ai/L test solution. No behavioral effects were seen at 100 mg ai/L with addition of 20 mg/L humic acid. NOEC for that test condition was 100% WAF of a 100 mg ai/L test solution. Mitigation of effects was thought to be due to adsorption of the test item to the humic acid.

With a water solubility of 86.4 mg/L those values would translate to the following approximate concentrations:
Without addition of humic acid:
LC50: 30.24 mg/L
NOEC: 21.6 mg/L
With addition of 10 mg/L of humic acid:
LC50: > 86.4 mg/L
NOEC: 43.2 mg/L
With addition of 20 mg/L of humic acid
LC50 > 86.4 mg/L
NOEC: 86.4 mg/L
Executive summary:

In a static acute toxicity test with Fathead Minnows (Pimephales promelas), the 96 hour LC50 values (lethality) was estimated by binomial probability to be 35% Water Accommodating Fraction prepared from 100 mg active ingredient test article/ L dilution water with 95% confidence intervals of 25 to 50% Water Accommodating Fraction of a 100 mg ai/L test solution. The No-Observed-Effect Concentration (NOEC) was determined to be 25% WAF of a 100 mg ai/L test solution.

To test a more real world situation, humic acid was added in low (10 mg/L) or high (20 mg//L) amounts to aquaria with the same test concentrations of test item. In both humic acid tests, no concentration of test item caused mortality in exposed fish. Thus, the 96 hour LC50 for test item plus 10 mg/l humic acid was greater than 100% WAF of a 100 mg ai/L test solution. From 72 hours on, fish under those conditions exposed to 100% WAF were lethargic. As no behavioral effects were seen in the remaining test concentrations, the 96 hour NOEC for that test condition was 50% WAF of a 100 mg ai/L test solution. No mortality or behavioral effects were seen at any test solution with 20 mg/L humic acid added. 96 hour LC50 for fish exposed to test item and 20 mg/L was > 100% WAF of a 100 mg ai/L test solution and the NOEC for those fish as 100% WAF of a 100 mg ai/L test solution.

Adsorption of the test item to humic acid was thought to explain the mitigation of effects. Strong adsorption was predicted by ECOSAR modelling of the test molecule.

Description of key information

An acute toxicity test has been performed with the test item under static condition using Fathead Minnows (Pimephales promelas). The test was conducted under OECD guidelines requirements (OECD guideline N° 203) and GLP.  Due to the limited solubility of the test substance in the test medium, the test was performed with a water accomodated fraction. 
With a water solubility of 86.4 mg/L those values would translate to the following approximate concentrations:
Without addition of humic acid:
LC50: 30.24 mg/L
NOEC: 21.6 mg/L
With addition of 10 mg/L of humic acid:
LC50: > 86.4 mg/L
NOEC: 43.2 mg/L
With addition of 20 mg/L of humic acid
LC50 > 86.4 mg/L
NOEC: 86.4 mg/L
The result showed a LC50 (96 hours) of approximately 30.24 mg/L (mortality based - nominal concentration) and a NOEC value of approximately 21.6 mg/L (behaviour based - nominal concentration). The study and results associated have been considered as valid and were used in the chemical safety assessment.

Key value for chemical safety assessment

LC50 for freshwater fish:
30.24 mg/L

Additional information

The above acute test has also been performed with humic acid (addition of 10 mg/L and 20 mg/L) in order to be closer to environmental conditions. In that case, it has been showen that the toxicity of the test item was reduced with higher concentrations of humic acid, due to adsorption phenomenon of the test item to the humic acid. Even if the latter case seems closer to the real environmental conditions, only experiment without the humic acid has been taken into consideration for the chemical safety report.