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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June 1996 to 04 September 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: U.S. EPA (1993)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Each of the five WAFs were prepared by combining the appropriate amount of test substance and dilution water in a mixing vessel equipped with a magnetic stiffer (the vortex extended from the surface approximately 25 % of the way to the bottom of the mixing vessel) and stirring these mixtures for approximately 20 hours, settling the mixtures for approximately four hours, and siphoning the water phase containing the WAF.

DILUTION WATER
Water used for acclimation of test organisms and for all toxicity testing was sterile enriched media adjusted to a target pH of 7.5 ± 0.1 with 0.1 N HCl prior to use.
Media used for the definitive toxicity test contained <10 mg/L particulate matter at the start of the toxicity test and 30 mg/L particulate matter at the end of the test (the final value resulted, at least in part, from the presence of algal cells).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata)
- Source: From a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin.
- Age of inoculum (at test initiation): The subsample of algae used to inoculate media at the start of the definitive test came from a seven-day-old culture.
- Identification: Identification of the culture organisms was verified using an appropriate taxonomic key

ACCLIMATION
- Acclimation period: 14 days
- Culturing media and conditions (same as test or not): Yes, the culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions before the definitive test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
Temperature of the incubator was measured and recorded daily (range 23.4 to 23.7 °C). The temperature was recorded continuously in a representative vessel of water incubated with the test vessels.
pH:
pH was determined in each test vessel at the beginning and end of the test (range 3.5 to 10.5). The pH measurement of one control vessel was not recorded at the end of the test.
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.3, 3.0, 33, 330 and 3300 mg/L.
Details on test conditions:
TEST SYSTEM
- Type: Capped with inverted glass beakers.
- Material, size, headspace, fill volume: 250 mL glass Erlenmeyer flasks that contained 100 mL of test solution (water depth was approximately 3 cm).
- Initial cells density: Approximately 10,000 cells per millilitre
- Control end cells density: Approximately 10,000 cells per millilitre
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: No
- Detailed composition if non-standard medium was used: See Table 1.
Media used for the definitive toxicity test contained <10 mg/L particulate matter at the start of the toxicity test and 30 mglL particulate matter at the end of the test (the final value resulted, at least in part, from the presence of algal cells).

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Yes. Water used for acclimation of test organisms and for all toxicity testing was sterile enriched media adjusted to a target pH of 7.5 ± 0.1 with 0.1 N HC1 prior to use.
- Photoperiod: A 24 hour light and 0 hour dark photoperiod.
- Light intensity and quality: Automatically maintained with cool-white fluorescent tights that provided a light intensity of 400 to 430 foot-candles
Test vessels were randomly arranged on a rotary shaker that was adjusted to 100 rpm and was located in an incubator during the test (a random numbers table was used to select the location of each vessel).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colours, flocculation, adherence of cells to test containers, or aggregation of cells were determined visually by means of direct microscopic examination with a haemocytometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 to 11
- Range finding study: Yes
- Test concentrations: Nominal 0 (control), 0.3, 3.0, 33, 330 and 3300 mg/L
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 33 to 330 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 33 to 330 mg/L
Details on results:
Insoluble material was observed at 24, 72, and 96 hours in vessels containing 330 mg/L and 3,300 mg/L test media (test solutions were turbid, and turbidity increased with concentration). The algal population grew well, resulting in an average of 1,508,000 cells/mL in the control after 96 hours. Water quality throughout the test was within acceptable limits.
The two highest concentrations of test substance significantly decreased the pH of test media at the beginning of the test. At test initiation, the pH of the 330 mg/L test solution ranged from 4.3 to 4.4, and the pH of the 3,300 mg/L test solution ranged from 3.9 to 4.0.

No effects (size differences, unusual cell shapes, colours, flocculations, adherence of cells to test containers, or aggregation of cells) were noted during the test.
The 96 hour NOEC is 33 mg/L. The calculated EC50 values and their associated 95 % confidence limits for algae exposed to the test material are as follows:

Calculated Using the Number of Cells/mL:
72 hour EC50 = 100 mg/L (95 % confidence interval = 33 to 330 mg/L)
96 hour EC50 = 93 mg/L (95 % confidence interval = 33 to 330 mg/L)

Calculated Using the Average Specific Growth Rate:
72 hour EC50 = 100 mg/L (95 % confidence interval = 33 to 330 mg/L)
96 hour EC50 = 100 mg/L (95 % confidence interval = 33 to 330 mg/L)

The determination of whether toxic effects were algistatic or algicidal was performed at the conclusion of the test; a 0.5 mL aliquot of test media from each 330 mg/L test material vessel was transferred to a flask containing 100 mL of fresh media and incubated under test conditions for 144 hours. Algae increased from <10,000 cell/mL to 578,000 cells/mL, indicating that the effect of the test material at the highest tested concentration was algistatic rather than algicidal.
Reported statistics and error estimates:
The average specific growth rate was calculated as the natural log of the number of cells/mL at 72 and 96 hours of exposure, minus the natural log of the number of cells/mL at 0 hours, divided by the hour of exposure. Results of the toxicity test were interpreted by standard statistical techniques. All calculations were performed using nominal concentrations of the test material with the number of cells/mL, then with the average specific growth rates.

The binomial/nonlinear interpolation method (Stephan, 1983) was used to calculate the 72 and 96 hour EC50 values. The no observed effect concentration (NOEC) is the highest concentration of test substance that allowed at least 90 % of control growth. (This definition of the NOEC differs slightly from the TSCA test guidelines).

Table 2: Cell Growth data

Nominal Concentration of Test Material
(mg/L)

Number of Cells per Millilitre
(Mean of replicate flasks)

0 hr

72 hr

96 hr

0 (control)

10,000

383,000

1,508,000

0.3

10,000

604,000

1,681,000

3.0

10,000

574,000

1,430,000

33

10,000

635,000

1,465,000

330

10,000

<10,000

<10,000

3,300

10,000

<10,000

<10,000

Table 3: Average Specific Growth Rate

Nominal Concentration of Test Material
(mg/L)

Average Specific Growth Rate

Percent of Control

72 hr

96 hr

72 hr

96 hr

0 (control)

0.051

0.052

-

-

0.3

0.057

0.053

112

102

3.0

0.056

0.052

110

100

33

0.058

0.052

114

100

330

0.000

0.000

0

0

3,300

0.000

0.000

0

0

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 96 h EC50 was 100 mg/L based on growth rate. The 96 h NOEC was determined to be 33 mg/L.
Executive summary:

The acute toxicity of the water accommodated fraction (WAF) of the test material to the freshwater alga, Selenastrum capricornutum, was investigated during a range-finding study; performed under GLP conditions and to the standardised guideline OECD 201.

The test, which was designed to establish the approximate toxicity of the test substance, was performed for 96 hours at 24 ± 1 °C under static conditions with a control (0 mg/L) and five concentrations of WAF (0.3, 3.0, 33, 330, and 3,300 mg/L).

The dilution water was sterile enriched media adjusted to a pH of 7.5 ± 0.1. The five WAFs were prepared by formulating individual concentrations of the test substance and dilution water in mixing vessels equipped with a magnetic stirrer, stirring the mixtures for approximately 20 hours, settling the mixtures for approximately four hours, and siphoning the water phase containing the WAF. During the mixing period, the vortex extended approximately 25 % of the distance from the water surface to the bottom of the mixing vessels.

Insoluble material was observed at 24, 72, and 96 hours in vessels containing 330 mg/L and 3,300 mg/L test media (test solutions were turbid, and turbidity increased with concentration). No other insoluble material was noted during the test.

Exposure of freshwater alga to the test material resulted in a 96 hour median effective concentration (EC50) of 93 mg/L (95 % confidence interval = 33 to 330 mg/L) when calculated using cells/mL, and 100 mg/L (95 % confidence interval = 33 to 330 mg/L) when calculated using average specific growth rate.

Under the conditions of this study, the 96 hour no observed effect concentration (NOEC) was determined to be 33 mg/L using both cells/mL and average specific growth rate.

Description of key information

The 96 h EC50 was 100 mg/L based on growth rate. The 96 h NOEC was determined to be 33 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
33 mg/L

Additional information

The acute toxicity of the water accommodated fraction (WAF) of the test material to the freshwater alga, Selenastrum capricornutum, was investigated during a range-finding study; performed under GLP conditions and to the standardised guideline OECD 201.

The test, which was designed to establish the approximate toxicity of the test substance, was performed for 96 hours at 24 ± 1 °C under static conditions with a control (0 mg/L) and five concentrations of WAF (0.3, 3.0, 33, 330, and 3,300 mg/L).

The dilution water was sterile enriched media adjusted to a pH of 7.5 ± 0.1. The five WAFs were prepared by formulating individual concentrations of the test substance and dilution water in mixing vessels equipped with a magnetic stirrer, stirring the mixtures for approximately 20 hours, settling the mixtures for approximately four hours, and siphoning the water phase containing the WAF. During the mixing period, the vortex extended approximately 25 % of the distance from the water surface to the bottom of the mixing vessels.

Insoluble material was observed at 24, 72, and 96 hours in vessels containing 330 mg/L and 3,300 mg/L test media (test solutions were turbid, and turbidity increased with concentration). No other insoluble material was noted during the test.

Exposure of freshwater alga to the test material resulted in a 96 hour median effective concentration (EC50) of 93 mg/L (95 % confidence interval = 33 to 330 mg/L) when calculated using cells/mL, and 100 mg/L (95 % confidence interval = 33 to 330 mg/L) when calculated using average specific growth rate.

Under the conditions of this study, the 96 hour no observed effect concentration (NOEC) was determined to be 33 mg/L using both cells/mL and average specific growth rate.