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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:
19 - 27 Nov 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011
Deviations:
yes
Remarks:
See section "Any other information on material and methods incl tables"
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
adopted 07. December 2015
Deviations:
yes
Remarks:
See section "Any other information on material and methods incl tables"
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz, Mainz, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and all test item solutions
- Sampling method: Samples were taken at test start and test end
- Sample storage conditions before analysis: Samples were frozen
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: A stock solution was prepared by mixing the nominal load of 100.0 mg/L with the corresponding amount of algal medium and shaking vigorously for 23 h. The resulting solution was filtrated through 0.45 μm cellulose filters. The lower treatments were prepared by dilution of this saturated solution with nutrient medium.
- Controls: Yes, algae medium only
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: SAG 86.81
- Source (laboratory, culture collection): Received from MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen; Jan 2016)
- Age of inoculum (at test initiation): 5 days (pre-culture)
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Acclimation period: 5 days (for the pre-culture)
- Culturing media and conditions (same as test or not): Not the same, stock culture was kept on a solid agar. For the pre-culture the same medium (OECD 201 medium) was used.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.2 – 23.2 °C
pH:
7.8 - 9.1 (Control)
7.8 - 10.1 (Test item concentrations)
Nominal and measured concentrations:
Nominal: Control, 1, 3.2, 10, 32 and 100 mg/L
Measured: measured concentrations are listed in table 5 - 6 in section "Any other information on results incl tables".
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: Glass flasks total volume 65 mL, 46 mL test solution, 15 mL headspace
- Initial cells density: 2400 cells/mL
- Control end cells density: 815737 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: OECD medium was used according to the OECD guideline 201, all solutions were sterilized before use.
- Culture medium different from test medium: Pre-culture and test were performed with the same medium
- Intervals of water quality measurement: pH was measured at test start and test end

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continous
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Cell numbers were determined with an electronic particle counter every 24 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: The concentrations to be tested are based on non-GLP pre-tests.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 56.58 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 56.58 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
18.11 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No, algae cells appeared in a normal shape through the whole test duration and for all test item concentrations and the control.
Others: the lowest test item concentration of 1 mg/L was excluded from the evaluation based on turbidity.
Results with reference substance (positive control):
72h ErC50: 0.79 mg/L (95% confidence interval 0.79 - 0.79 mg/L)
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1. statistical calculation using ToxRat® Professional 3.2.1 (based on nominal concentrations) and statistical calculation using ToxRat® Professional 3.2.1 (based on measured concentrations).

Table 1: Biological Results of the Test Item (based on measured concentrations)

Parameter

Value

95% confidence interval

NOEC (Growth Rate) 72 h

18.11 μg/L

 --

NOEC (Yield) 72 h

 18.11 μg/L

 --

LOEC (Growth Rate) 72 h

 56.58 μg/L

 --

LOEC (Yield) 72 h

 56.58 μg/L

--

72h ErC10

> 56.58 μg/L

--

72h EyC10

 15.16 μg/L

 2.51 – 91.75 μg/L

72h ErC50

 > 56.58 μg/L

 --

72h EyC50

> 56.58 μg/L

 --

 

Table 2: Biological Results of the Test Item (based on nominal concentrations)

Parameter

 Value

 95% confidence interval

NOEC (Growth Rate) 72 h

 32.00 mg/L

--

NOEC (Yield) 72 h

32.00 mg/L

 --

LOEC (Growth Rate) 72 h

 100.00 mg/L

--

LOEC (Yield) 72 h

100.00 mg/L

--

72h ErC10

> 100 mg/L

 --

72h EyC10

26.79 mg/L

 4.43 – 162.18 mg/L

72h ErC50

> 100 mg/L

--

72h EyC50

> 100 mg/L

 --

 

Table 3: Inhibition Values

Nominal concentration in mg/L

 % Inhibition

 

 

Growth Rate (0-72h)

Yield (0-72h)

Blank control

0

0

1*

11.85

 49.74

3.2

 -1.7

-10.0

10

 -0.9

 -5.1

32

 2.6

 14.5

100

8.2

37.3

Negative inhibition values indicate a stimulation of algal growth compared to the blank control. Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation.

* values calculated by Microsoft Excel®, not by ToxRat®

 

Table 4: Cell Numbers calculated

Nominal Conc.

Replicate

Cell Number/mL

[mg/L]

 

0 h

24 h

48 h

72 h

Blank control

1

2400

19440

161800

815900

 

2

2400

21140

152620

755260

 

3

2400

18120

175740

928160

 

4

2400

18120

152960

860020

 

5

2400

18660

160920

828940

 

6

2400

16500

133900

706140

 

Mean

2400

18663

156323

815737

 

SD

0

1550

13827

78030

1

1

2400

14760

116060

475020

 

2

2400

15680

115240

417220

 

3

2400

14000

79860

341260

 

Mean

2400

14813

103720

411167

 

SD

0

841

20667

67085

3.2

1

2400

18360

170360

919060

 

2

2400

19260

163940

874420

 

3

2400

19620

176140

898040

 

Mean

2400

19080

170147

897173

 

SD

0

649

6103

22333

10

1

2400

17920

160840

864980

 

2

2400

19980

160320

940620

 

3

2400

18460

151380

766140

 

Mean

2400

18787

157513

857247

 

SD

0

1068

5318

87497

32

1

2400

15920

119660

675140

 

2

2400

17460

162200

728120

 

3

2400

14880

137700

689480

 

Mean

2400

16087

139853

697580

 

SD

0

1298

21352

27403

100

1

2400

14620

120080

637200

 

2

2400

12400

99600

510440

 

3

2400

13560

85960

390520

 

Mean

2400

13527

101880

512720

 

SD

0

1110

17174

123356

SD = Standard Deviation

Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation

Analytical Results:

Table 5: Measured Concentrations with algae

Nominal Concentration Test Item mg/L

Measured concentration test item μg/L

 

 

 

 

t = 0 h

 t = 24 h

 t = 48 h

 t = 72 h

Blank control

--

--

--

<LOD

1

--

<LOD

<LOD

<LOD

3.2

--

<LOD

<LOD

<LOD

10

--

<LOD

<LOD

<LOD

32

--

10.39

3.50

<LOD

100

--

49.90*

40.02

31.90

LOD / LOQ (Limit of detection / quantification) = 1.00 μg/L (as the lowest calibration level)

Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation

* 8-fold diluted instead of 2-fold like the other values

 

Table Measured Concentrations without algae

Nominal Concentration Test Item

Measured concentration test item μg/L

mg/L

t = 0 h

 t = 24 h

 t = 48 h

 t = 72 h

Blank control

< LOD

--

--

--

1

< LOD

< LOD

< LOD

< LOD

3.2

1.24

< LOD

< LOD

< LOD

10

5.81

< LOD

< LOD

< LOD

32

24.46

9.82

5.94

3.89

100

96.93*

55.74*

45.72

41.50

100 (additional)

82.00

--

--

--

LOD / LOQ (Limit of detection / quantification) = 1.00 μg/L (as the lowest calibration level)

Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation

* 8-fold diluted instead of 2-fold like the other values

Table 6: Geometric Mean of Measured Concentrations and Calculated Concentrations

Nominal Concentration Test Item

Geometric Mean

 Calculated Concentrations

mg/L

 μg/L

 μg/L

Blank control

 -

- --

1

--

0.57

3.2

 --

 1.81

10

 --

5.66

32

--

 18.11

100

 56.58

 56.58

Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation

Table 7: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

340

Yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

14%

Yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

2%

Yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

EC50 (72h) >56.58 µg/L (Desmodesmus subspicatus, OECD 201, mean measured concentrations)

EC10 (72h) >56.58 µg/L (Desmodesmus subspicatus, OECD 201, mean measured concentrations)

Key value for chemical safety assessment

Additional information

One experimental study is available investigating the toxicity of 4-(octadecylamino)-4-oxoisocrotonic acid (CAS 3077-27-8) to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) using the freshwater algae Desmodesmus subspicatus (LAUS GmbH, 2018b). The test was carried out with test item concentrations of 1, 3.2, 10, 32 and 100 mg/L for 72 h. The test solution was prepared by adding appropriate amounts of test item to test medium, followed by intensive stirring and filtration. During the study the actual concentrations in the test medium was measured by HPLC/MS. The measured concentration in the freshly prepared solutions was between 1.24 μg/L and 96.93 μg/L and in the aged test solutions between 3.89 μg/L and 41.5 μg/L, respectively. Therefore the results are given based on the geometric mean of the measured concentrations. Hence the EC50 (72 h) was determined to be > 100 mg/L (nominal) and > 56.58 μg/L (geom. mean measured concentration), respectively. Statistically significant effects were determined at the highest test item concentration of 56.58 µg/L based on growth rate. Hence the NOEC was determined to be at 18.11 µg/L. Due to a low inhibition of 8% at 56.58 µg/L the EC10 was used as toxicological endpoint since effects below 10 % compared to the control are generally not considered to be ecotoxicologically relevant. According to the Guidance on Application of CLP criteria (Version 5.0, July 2017) the preference is given to EC10 to be used for classification and risk assessment. Following this strategy the EC10 and EC50 were used as toxicological endpoints. The measured water solubility for the test item was determined to be 5.9 mg/L at 25 °C, pH 5. The discrepancy between the low recovery in the test solutions and the water solubility may have been caused by the different test media, pH-values and temperature.