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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:
1 to 5 february 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictons. The measured concentrations were outside the 80-120% range. Therefore the results were expressed as means of measured concentrations.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control and vehicle control), 0.65, 1.3, 2.5, 5, 10
- Sampling method: at 0-hour, 10 mL samples were collected from each parent solution. At 96-hours, composite samples were collected from each test substance and control treatment. The abiotic control flask was also sampled at this time. The 96-hours samples were centrifuged in glass culture tubes to remove the algal biomass. Samples at concentration below 0.300 mg/L were diluted with acetonitrile to a final concentration of 50%; samples above 0.65 mg/L were diluted in 80:20 acetonitrile: ABC reagent water (v:v) such that the concentration of analyte in the final extracts was within the range of the standard reference curve. Quality control fortifications samples (spikes) were prepared by spiking 10.0 mL of control water with Super Radjanol. Spikes were prepared at concentrations braketing the range of nominal concentrations of the test substance treatments.


Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMF
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 µL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no evidence of undissolved material
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornatum
- Strain: 99-A5-SEL
- Source (laboratory, culture collection): Departement of Botany, Culture collection of algae, University of Texas, Austin
- Age of inoculum (at test initiation): 6 days old
- Method of cultivation: the parent stock of algae were received growing on an agar slant contained in a culture tube and was assigned ABC lot 98-1648-4. Periodically, new Selenastrum capricornatum culture was initiated using a portion of this parent stock or cloned for an existing culture derived from the parent stock in 100 mL of autoclaved culture medium.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
no post exposure obsrvation period.
Test temperature:
22.4 to 22.9 °C at test initiation
23.4 to 24.3 °C at test termination
The test temperature was within the range specified in the protocol (24 ± 2°C)
pH:
7.5 to 7.6 at test initiation
8.4 to 8.7 at test termination
Nominal and measured concentrations:
Nominal concentrations: 0, 0.65, 1.3, 2.5, 5, 10 mg a.i/L
Measured concentrations (arithmetic means of T=0 and T=96h) : 0, 0.44, 0.8, 1.2, 2.6, 3.6
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml Erlenmeyer flasks fitted with foam stoppers
- Type (delete if not applicable): closed
- Continuous agitation, agitation rate = 100 rpm
- Initial cells density: 1x104 cells/mL
- Control end cells density: 1.7x106 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: prepared in ABC reagent water which is defined as reverse osmosis/deionized water passed through carbon, ion exchange and organic adsorption cartridges and filtered through a 0.2 µm hollow fiber final filter to produce a 16-18 megaohms.cm water
- Metals: < 0.1 mg/L
- Pesticides: < 4 µL
- Chlorine: < 0.025 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustement
- Light intensity and quality: continuous cool white fluorescent lighting (4000 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: hematocytometer and Olympus Model BH-2 microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0 (control and vehicle control), 0.1, 1, 10, 20 ,40 mg a.i/L
- Results used to determine the conditions for the definitive study: After 96 hours of incubation, percent decrease in cell density as compared to the vehicle control was 10, 76, 82 and 87% at 1, 10, 20 and 40 mg/L respectively. No cells were counted at 0.1 mg/L indicating a possible poor inoculation of this flask.

See table 1: Measured concentrations of Super Radjanol in media sampled during a 96-hour static test with the freshwater green alga, Selenastrum capricornatum
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 2.1 - 2.8 mg/L
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.44 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
Table 1: Mean cell numbers (as determined by Hemacytometer) during a 96-hour continuous exposure of the freshwater green alga, Selenastrum capricornatum, to Super Radjanol under static test conditions
Table 2: Calculated EC50 values based on mean measured concentrations for the freshwater green alga, Selenastrum capricornatum, exposed to Super Radjanol under static conditions
Table 3: Daily cell counts of the freshwater green alga, Selenastrum capricornatum, during a 96-hour static exposure to Super Radjanol
Results with reference substance (positive control):
no positive control

Table 1: Mean cell numbers (as determined by Hemacytometer) during a 96-hour continuous exposure of the freshwater green alga, Selenastrum capricornatum, to the test item under static test conditions

 

Measured concentrations (mg/L)

Mean cell number (x104) /mLa

Percent inhibitionb

Day 0

Day 1

Day 2

Day 3

Day 4

Control

1.11

2.00

8.62

27.7

172

--

Vehicle control

1.00

1.63

7.64

25.6

146

--

0.44

--

1.30

7.41

26.7

128

12

0.80

--

1.07

4.59

28.2

113

23d

1.2

--

1.15

4.41

26.7

94.0

36d

2.6

--

1.18

3.41

13.1

67.3

54d

3.6

--

0.96

2.85

7.61

64.2c

56d

a: Values are mean of triplicate test chambers.

b: Percent inhibition is determined against the vehicle control at 96 -hours as: %inhibition = ((cells/mL of vehicle control - cells/mL of treatment)/ (cells/mL of vehicle control)) x 100

c: Replicate C was an outlier and was not included in the mean calculation

d: Significant reduction in growth compared to the vehicle control (α = 0.05)

Table 2: Calculated EC50values based on mean measured concentrations for the freshwater green alga,Selenastrum capricornatum, exposed to the test item under static conditions

EC50time period

EC50value (mg a.i/L)

95% confidence limit (mg a.i/L)

RMSEb

R2

24

26a

0-191

18.2

6.80

48

1.7

0.75-2.7

16.3

37.5

96

2.7

2.2-3.2

27.5

61.0

120

2.5

2.1-2.8

5.93

90.1

a: greater than the highest concentration tested

b: Root Mean Square Error. Smaller values indicate a better fit of the regression line. The minimum possible value is 0.0

 

Table 3: Daily cell counts of the freshwater green alga, Selenastrum capricornutum, during a 96-hour static exposure to the test item

Mean measured concentrations (mg/L)

REP

Cells per mL (x104)

0-hour

24-hour

48-hour

72-hour

96-hour

Control

A

1.11

1.78

8.00

30.0

172

B

1.00

2.11

7.67

34.0

173

C

1.22

2.11

10.2

19.0

171

Vehicle control

A

1.00

1.56

3.89

27.8

157

B

0.89

2.11

10.7

10.8

139

C

1.11

1.22

8.33

31.0

141

0.437

A

--

1.78

5.22

19.5

133

B

--

0.67

5.67

25.5

116

C

--

1.44

5.33

35.0

134

0.796

A

--

1.11

2.11

39.5

106

B

--

0.89

7.56

24.3

111

C

--

1.22

4.11

20.8

123

1.25

A

--

1.11

4.67

18.8

84.8

B

--

1.33

3.22

24.0

97.5

C

--

1.00

5.33

37.3

99.8

2.6

A

--

1.33

3.56

11.3

65.0

B

--

0.89

3.33

13.5

59.5

C

--

1.33

3.33

14.5

77.5

3.59

A

--

1.00

3.67

13.5

66.8

B

--

1.22

3.22

8.33

61.5

C

--

0.76

1.67

1.00

8.00

 

Validity criteria fulfilled:
yes
Conclusions:
The 96-hour EC50 was estimated to be 2.5 mg a.i/L with 95% confidence limits of 2.1 and 2.8 mg/L
The NOEC was 0.44 mg a.i/L
Executive summary:

In a 96 hour acute toxicity study, the cultures of Selenastrum capricornatum were exposed to the test item at measured concentrations of 0 (control and vehicle control), 0.44, 0.80, 1.2, 2.6, 3.6 mg a.i./L under static conditions in accordance with the EPA OPPTS 850.5400 guideline. The EC50  and NOEC values based on cell density were 2.5 and 0.44 mg a.i./L, respectively. The % growth inhibition in the treated algal culture as compared to the vehicle control ranged from 12 to 56.

 

There were no compound-related phytotoxic effects.

 

This toxicity study is considered as acceptable and satisfies with the guidelines requirements for freshwater green alga toxicity study.

 

Results Synopsis

 

Test Organism: Freshwater green alga, Selenastrum capricornatum

Test Type (Flowthrough, Static, Static Renewal): static

 

96-hr EC50: 2.5 mg a.i./L               95% C.I.: 2.1 to 2.8 mg a.i./L

96-hr NOEC: 0.44mg a.i./L

                  

Endpoint(s) Effected: inhibition growth

Description of key information

The substance exhibits an 96h-EC50 for freshwater algae of 2.5 mg/L and a 96h-NOEC of 0.44 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
2.5 mg/L
EC10 or NOEC for freshwater algae:
0.44 mg/L

Additional information

One experimental study and one QSAR are available to determine the toxicity to aquatic algae.

Malorin, 1999

In a 96-hour acute toxicity study, cultures of Selenastrum capricornatum were exposed to Super Radjanol at nominal concentrations of 0, 0.65, 1.3, 2.5, 5.0, 10.0 mg/L under static conditions in accordance with the EPA OPPTS 850.5400 guideline. The concentrations of the test item (with and without algae) were determined by HPLC at the start and at the end of the test. The NOEC and EC50 values based on growth rate were 0.44 and 2.5 mg/L, respectively.

This toxicity study is classified as acceptable and satisfies the guideline requirement for growth inhibition of algae study.

Thomas, 2015

A QSAR model was used to predict the inhibition of growth to algae of the test item 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-(2E)-buten-1-ol. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (1), referenced as Method C.3 of Commission Regulation No. 440/2008 (2). The first criterion predicted was the NOECr (No Observed Effect Concentration for specific growth rate), a tested concentration which is expected to cause no effect on intrinsic rate of growth of the test system within a period of 72 hours. The other predicted criterion was the ErC50(Median Effective Concentration for specific growth rate), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of growth of the test system within a period of 72 hours.

The QSAR models are based on validated data for a training set of 40 chemicals derived from 72-hour ErC50and 31 chemicals derived from 72-hour NOECr test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The 72h-ErC50 of 2-ethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)but-2-en-1-ol to algae was predicted as 1.4 mg/L.

The 72h-NOErC of 2-ethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)but-2-en-1-ol to algae was predicted as 0.49 mg/L.

This toxicity study is considered acceptable and satisfies the guideline requirements for growth inhibition of algae study.