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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
A stock solution was prepared from 13.7 mg Diuron in 2.0 L deionised water, 35 min ultrasonication and 35 min stirring.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: SAG 86/81
- Source (laboratory, culture collection): laboratory culture
- Method of cultivation: Semi continuous cultivation under 16 hours photoperiod, at 20 °C, autoclaved nutrient solution according to Bringmann and Kühn (1980).

ACCLIMATION
- Acclimation period: Pre culture to ensure exponential growth of the test cultures
- Culturing media: OECD TG 201 medium, same as test
- Initial cell concentration: 1.0 x 10 4 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
Samples counted after 24, 48, 72 and 96 hours
Hardness:
not given
Test temperature:
23 ± 2 °C
pH:
Initial: 7.71 – 7.96, at termination: 7.39 – 9.09
Dissolved oxygen:
not given
Salinity:
not given
Nominal and measured concentrations:
Initial concentrations of test substance Control, 0.0001, 0.00032, 0.00056, 0.001, 0.0018, 0.0032, 0.0056, 0.01 and 0.032 mg Diuron/L
The average of mean measured concentrations in the main test was 96.7 % of the nominal
Main test: For 0.00010 0.0001 mg Diuron/L
For 0.00032 0.00033 mg Diuron/L
For 0.00056 0.00056 mg Diuron/L
For 0.0010 0.0009 mg Diuron/L
For 0.0018 0.0017 mg Diuron/L
For 0.0032 0.0023 mg Diuron/L
For 0.0056 0.0060 mg Diuron/L
For 0.010 0.011 mg Diuron/L
For 0.032 0.030 mg Diuron/L

Details on test conditions:
TEST SYSTEM
- Test vessel: Incubator with illumination from side lamps
- Type: open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks, 150 mL test medium
- Aeration: None, however, Intermittent turning for suspending algae
- Initial cells density: 1.0 x 104 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: See Table 1.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous
- Light intensity and quality: 8000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: photometric evaluation of extinction

TEST CONCENTRATIONS
- Test concentrations:
Nominal: Control, 0.0001, 0.00032, 0.00056, 0.001, 0.0018, 0.0032, 0.0056, 0.01 and 0.032 mg Diuron/L
The average of mean measured concentrations in the main test was 96.7 % of the nominal



Reference substance (positive control):
yes
Remarks:
K2Cr2O7: at concentrations of 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3.2 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
22 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
22 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
7.9 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control: yes (see table 3)
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50 (72h): 1.38 mg/L
- EbC50 (72h): 0.59 mg/L

The EbC50 of 0.59 mg/L falls within the collaborative study range of 0.20 – 0.75 mg/L
The ErC50 of 1.38 mg/L agrees with the collaborative study range of 0.60 – 1.03 mg/L

Reported statistics and error estimates:
The ErC50 and EbC50 values were determined by a probit analysis by the method of maximum likelihood.
The NOEC was determined by a Dunnett’s Test (analysis of variance).

Table 2:           Cell concentration data

Test-Substance

Concentration
(nominal)

[mg/L]

Cell densities (mean values)

(algae 104cells/mL)

Measured

Percent inhibition

Growth rate

Biomass

 

0 h

24 h

48 h

72 h

96 h

72 h

96 h

72 h

96 h

Control

1.0

4.59

12.49

51.35

125.85

-

-

-

-

0.00010

1.0

4.45

15.11

56.10

125.43

-2.2

0.1

-12.1

-5.5

0.00032

1.0

4.32

11.02

53.79

115.06

-1.2

1.9

1.3

3.7

0.00056

1.0

5.20

13.82

48.35

118.96

1.5

1.2

-1.1

3.5

0.0010

1.0

4.66

15.58

50.20

133.47

0.6

-1.2

-6.4

-4.6

0.0018

1.0

5.60

13.34

33.71

113.56

10.7

2.1

17.3

17.1

0.0032

1.0

5.53

12.65

47.39

118.27

0.2

1.3

2.2

5.2

0.0056

1.0

2.97*

6.68*

25.10*

72.30*

18.2

11.5

51.1

47.3

0.010

1.0

3.17*

7.36*

18.04*

39.98*

26.6

23.7

57.6

64.8

0.032

1.0

2.76*

4.72*

4.93*

6.08*

59.5

62.7

81.5

90.7

Temperature [°C]

n.d.

n.d.

n.d.

n.d.

21.8 – 22.6

 

pH

7.71 – 7.96

7.43 – 8.01

7.52 – 7.93

8.02 – 8.39

7.39 – 9.09

 

*significant difference to control (p = 0.05).

Table 3: Validity criteria for the growth inhibition test on algae according to OECD Guideline 201

 

Fulfilled

Not fulfilled

Cell concentration in control cultures increased at least by a factor of 16 within 3 days 

x

-

Concentration of test substance³80% of initial concentration during test

x

-

 

Executive summary:

A short-term acute toxicity test, in accordance with the prevailing OECD testing guidelines (OECD 201) and GLP, is available for this substance.

Based on measured concentrations , the 72h EC50 (growth rate) was calculated to be 22 µg/L test item, while the 72h EC50 (biomass) was calculated to be 9 µg/L. The NOEC (growth rate) was calculated to be 3.2 µg/L.

Therefore the test item (Diuron) was determined to be acutely toxic to Scenedesmus subspicatus.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
modified for use with Anabena flos-aquae
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
A stock solution of 10 mg/L was prepared in purified water by ultrasonication followed by 3 hours stirring.
Test organisms (species):
Anabaena flos-aquae
Details on test organisms:
TEST ORGANISM
- Common name: blue-green alga (cyanophyta)
- Strain: B 30.87
- Source: Culture Collection of algae cultures, Pflanzenphysiologisches Institut der Universität Göttingen, Germany

ACCLIMATION
- Acclimation period: test algae taken from an exponentially growing culture set up 3 days prior to start of study
- Culturing media and conditions: Same as test
- Initial cell concentration 1.0 x 104 cells/mL
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/L CaCO3
Test temperature:
24 °C
pH:
Start: 7.9 – 8.1, termination: 8.1 – 9.2
Dissolved oxygen:
not given
Salinity:
not given
Nominal and measured concentrations:
Nominal: Control, 0.0001, 0.0032, 0.0010, 0.032 and 0.1 mg Diuron/L
The mean measured concentrations in the main test ranged from 95 % to 104 % of the nominal
Main test: For 0.01 0.00101 mg Diuron/L
For 0.032 0.031 mg Diuron/L
For 0.100 0.103 mg Diuron/L
Details on test conditions:
TEST SYSTEM
- Test vessel: water bath with light source from above
- Type: open
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks
- No. of organisms per vessel: 1.0 x 104 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: See Table 1
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- pH: Start: 7.9 – 8.1, termination: 8.1 – 9.2
- Photoperiod: Continuous
- Light intensity and quality: Mean 7600 lux
- Temperature: 24 °C

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
Nominal: Control, 0.0001, 0.0032, 0.0010, 0.032 and 0.1 mg Diuron/L
The mean measured concentrations in the main test ranged from 95 % to 104 % of the nominal
Main test: For 0.01 0.00101 mg Diuron/L
For 0.032 0.031 mg Diuron/L
For 0.100 0.103 mg Diuron/L
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
10.1 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30.9 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
232 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL
Details on results:
- Exponential growth in the control: yes
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Growth rate and biomass both significantly inhibited at concentreations of test substance of 0.032 and 0.1 mg/L (p = 0.05, Dunnett-test)

Table 2          Cell concentration data of the main test

Test-Substance

Concentration
(nominal)

[mg/L]

Cell densities (mean values)

(algae 104cells/mL)

Measured

Percent inhibition

Growth rate

Biomass

 

0 h

24 h

48 h

72 h

72 h

72 h

Control

1.0

2.71

5.4

16.53

-

-

0.001

1.0

1.83

5.65

17.30

-1.6

1.7

0.0032

1.0

2.63

5.40

16.08

1.0

2.2

0.010

1.0

2.53

5.45

16.20

1.0

2.1

0.032

1.0

1.82

1.75

2.32

70.4*

84.0*

0.1

1.0

1.68

1.62

1.58

83.7*

88.5*

Temperature [°C]

24

24

24

24

 

pH

7.9 – 8.1

-

-

8.1 – 9.2

 

*significant difference to control (p =0.05).

Table 3.   Validity criteria for the growth inhibition test on algae according to OECD Guideline 201

 

Fulfilled

Not fulfilled

Cell concentration in control cultures increased at least by a factor of 16 within 3 days 

x

-

Coefficient of variation of average growth in replicate control cultures below 10 %

x

-

Coefficient of variation of daily growth rates in the controls below 35 %

x

-

Concentration of test substance³80% of initial concentration during test

x

-

 

Executive summary:

A short-term, acute toxicity test is available for this substance. Conducted in accordance with the prevailing OECD testing guidelines (OECD 201) and GLP, modified for use with the cyanophyta, Anabaena flos-aquae, as the test organism in this case.

Based on measured concentrations , the 72h EC50 (growth rate) was calculated to be 30.9 µg/L test item, while the 72h EC50 (biomass) was calculated to be 232 µ.g/L. The NOEC (growth rate) was calculated to be 10 µg/L.

Therefore the test item (Diuron) was proved to be acutely toxic to Anabaena flos-aquae.

Description of key information

Diuron is toxic to aquatic algae

Key value for chemical safety assessment

Additional information

Three toxicity tests with aquatic algae and cyanobacteria, all according to OECD guidelines (OECD 201 and 221) and GLP, are available for Diuron. A key study using the green alga, Scenedesmus subspicatus, calculated an (72 h) EC50 (growth) of 22 µg/L with a NOEC value of 3.2 µg/L (Heimbach, 1991). Since this is a standard species for acute toxicity testing in algae then these values are used as evidence of Diuron causing acute toxicity to aquatic plant species. Two supporting studies also determined EC50 and NOEC values of similar concentrations. The (72 h) EC50 ( growth) and NOEC values for Anabaena flos-aquae (cyanophyta) were 30.9 µg/L and 10 µg/L respectively (Memmert, 1998).