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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Oct 2019 - 10 Feb 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted 23 March 2006, Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test item concentration and conntrol
- Sampling method: At the start of the test (0 hours), 8 mL samples of freshly prepared test media were taken from the control and the test media preparation vessels for chemical analysis. At 24, 48 and 72 hours, 8 mL samples were also taken for chemical analysis from the pooled old test media from the test and control groups. In each case triplicate samples were taken, one for chemical analysis and two as ‘back-up’ samples should further analysis be required.



Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test, amounts (0.63, 1.29, 2.47, 5.09 and 10.07 mg) of test substance were separately added to 1000 mL of OECD medium. The preparations were stirred for ca 24 hours followed by a ca 1 hour settlement period after which the aqueous phase of each preparation was removed.
- Controls: The control was prepared by adding OECD medium only to the vessels.
- Test concentration separation factor: 2.0
- Evidence of undissolved material: The freshly prepared (new) test media for the control and test concentrations at the start of the test were observed to be colourless solutions.After 72 hours the test solution appeared as a homogenous, hazy dispersion of algae cells
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: Raphidocelis subcapitata (Strain 278/4; formerly known as Pseudokirchneriella subcapitata)
- Source: originally obtained from the Culture Collection of Algae and Protozoa (CCAP), Oban, UK
- Age of inoculum (at test initiation): Culture was incubated for ca 72 hours prior to the start of each test.
- Method of cultivation: Prior to the definitive test, two starter cultures were prepared from a current master culture at an inoculum concentration of 1 x 10E^04 cells/mL (1 mL of the master culture added to 100 mL of OECD media); incubation conditions were the same as used in the test

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.0 – 22.4 °C
pH:
Control: 7.82 - 9.52
Test item concentrations: 7.84 - 8.78
Nominal and measured concentrations:
Control, 0.625, 1.25, 2.5, 5.0 and 10 mg/L (nominal)
Control, 0.549, 0.762, 1.15, 1.67 and 1.68 mg/L (geometric mean measured conc)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: Sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks, filled with 100 mL of test or control medium, headspace of 150 mL, flasks were capped with foam bungs.
- Initial cells density: starting alga cell concentration of 1 x 10E^04 cells/mL
- Control end cells density: 134 x 10E^04 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: Stock solutions for Alga culture medium contained various nutrients which were prepared with reverse osmosis (RO) water. Aliquots of each of the stock solutions were then added to RO water. The stock solutions/alga culture medium were sterilised by autoclaving or filtering (0.2 m filter pore size) as appropriate.
- Metals: FeCl3.6H2O: 0.064 mg/L
- Ca/mg ratio: MgCl2.6H2O: 12 mg/L; CaCl2.2H2O: 18 mg/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: ph: at test start and test end; Temperature: continously (controlled incubator)

OTHER TEST CONDITIONS

- Adjustment of pH: No
- Photoperiod: Continously
- Light intensity and quality: Light intensity at 0 hours: 5890 – 7220 Lux; Light intensity at 72 hours: 5740 – 7280 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The cell density of the vial contents was then determined using a particle counter (Z2 Coulter Counter®).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study
- Test concentrations: Control; 0.1; 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: 0%, -26%, 1% and 95% (inhibition of growth rate); based on these results the test item concentrations for the main test were prepared
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.68 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits
Remarks:
0.805 – 1.34 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.762 mg/L
Nominal / measured:
meas. (geom. mean)
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):
- Unusual cell shape: after 72 h the Control, 0.625 and 1.25 mg/L: algae cells were bright green colour, sickle shaped; at 2.5 mg/L:Cells were bright green, mainly sickle shaped with a few flat cells; at 5.0 and 10 mg/L: cells were bright green, mainly sickle shaped with a few flat cells,
- Colour differences: No
- Flocculation: some flocculation of cells at 5 and 10 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.84 mg/L for growth rate
- Other: NOEC for growth rate was determined to be at 0.32 mg/L
Reported statistics and error estimates:
Statistical analysis was performed using the CETIS program v 1.8.6.8.
Average specific growth rate: 0-24 hour, 0-48 hour and 0-72 hour. In addition, 24-48 hour and 48-72 hour growth rate was also assessed. The no observed effect concentrations (NOEC) were determined using the following tests;
Dunnett Multiple Comparison Test: 0-24, 24-48 and 48-72 hours average specific growth rate. Williams Multiple Comparison Test: 0-48 and 0-72-hour average specific growth rate and 72-hour yield.
The EC10, EC20 and EC50 values for the 72-hour final yield, the 0-24, 0-48, 0-72, 24-48 and 48-72 hour average specific growth rate (μ) time intervals were estimated using linear interpolation.
To distinguish between ECx values, estimated using final yield and growth rates, the symbols EyCx and ErCx were used, respectively.

For presentation purposes, some numerical data may have been rounded; therefore, manual recalculation may result in slightly different values to those presented in the results section.

Biological Results

Table 1: Inhibition of growth rate in %

Nominal concentration (mg/L)

Geometric mean measured concentration
(mg/L)

Reduction in average specific growth
 rates relative to the control (%)

0-24
hours

0-48 hours

0-72
hours

24-48 hours

48-72 hours

Control

Control

-

-

-

-

-

0.625

0.549

0

-3

2

-5

10

1.25

0.762

-2

5

3

11

0

2.5

1.15

18

6

12

-4

22

5.0

1.67

44

32

27

22

19

10

1.68

49

33

33

18

35

-

Not applicable

Negative percentage inhibition values relative to the control represent an increase in growth relative to the control

Table 2: Endpints

Parameter

Geometric mean measured concentration (mg/L)

72-Hour

Yield

EyC10

0.645 (NC – 0.950)

EyC20

0.832 (NC – 0.997)

EyC50

1.23 (0.941 – 1.46)

NOEC

0.762

Growth Rate

ErC10

1.05 (0.805 – 1.34)

ErC20

1.40 (1.19 – 1.55)

ErC50

>1.68

NOEC

0.762

 (x – x) = 95% confidence limits; NC = Not calculated

Table 3: Cell Growth of Raphidocelis subcapitata during the Definitive Test

Nominal concentration (mg/L)

Geometric mean measured concentration
(mg/L)

Cell Concentration
(cells/mL)

Mean Cell Concentration
(cells x 104/ mL)

24-hour

48-hour

72-hour

24-hour

48-hour

72-hour

Control

Control

18010*

79000*

366220*

4.33

21.9

134

47280

233140

1225760

45460

225940

1490820

39000

119920

1167140

38690

258540

1289400

46250

255060

1504520

0.625

0.549

32780

171560

810560

4.40

23.4

125

45730

252220

1441640

53560

277440

1490700

1.25

0.762

50950

192120

1126200

4.45

18.6

115

39430

220100

1146300

43190

144480

1181920

2.5

1.15

25320

136100

721060

3.39

17.9

73.8

34830

162800

592460

41500

236960

899880

5.0

1.67

22200

74740

365480

2.29

7.91

35.2

20730

68920

303120

25800

93640

388620

10

1.68

18500

77040

303120

2.11

7.78

26.1

21080

69500

210500

23700

86900

270440

 * Replicate not included in results calculation as shown to be a statistical outlier (Grubbs Extreme Value test)

Analytical Results of the main test

Table 4: Chemical analysis during the Definitive Test

Nominal concentration (mg/L)

Measured concentration (mg/L)

Geometric mean measured concentration(mg/L)

0 hours (new media)

24 hours (old media)

48 hours (old media)

72 hours (old media)

Control

<LOQ

<LOQ

<LOQ

<LOQ

<LOQ

0.625

0.731 (117)

0.650 (104)

0.579 (93)

0.330 (53)

0.549 (88)

1.25

1.05 (84)

0.920 (74)

0.751 (60)

0.464 (37)

0.762 (61)

2.5

1.76 (70)

1.46 (58)

1.07 (43)

0.631 (25)

1.15 (46)

5.0

2.74 (55)

2.01 (40)

1.43 (29)

0.990 (20)

1.67 (33)

10

2.68 (27)

2.32 (23)

1.45 (15)

0.877 (9)

1.68 (17)

LOQ = Limit of quantification of the analytical method (0.001 mg/L)
Values in brackets represent % nominal value. However, it should be noted that the nominal concentrations of 5.0 and 10 mg/L were in excess of the solubility of the test substance

Table 1: Validity criteria for OECD 201 (2006)

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.

133

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%

15.5

 

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%.

2.35

 

Summary of results from the range-finding test

 

Specific growth rate and % inhibition relative to control

Nominal concentration
(mg/L)

0 to 72 hours

(x 10-2 cells/mL)

% Inhibition relative to control

Control

3.971

NA

0.10

5.022

-26

1.0

3.947

1

10

0.209

95

NA – Not applicable
Negative percentage inhibition values relative to the control are indicative of stimulated growth and are not considered to be an adverse effect

The biological results from the range-finding test differed from those obtained in the definitive test in that 95% reduction in growth rate was observed at the highest concentration in the range-finding test compared to 33% observed in the definitive test. The highest concentration was prepared from an initial 10 mg/L loading rate which was in excess of the solubility of the test substance in test media. The measured concentration at the highest concentration during the range-finding test was ca 30% greater than that observed during the definitive test (this is not unusual when the test substance is prepared from an excess of material) which could have resulted in increased growth inhibition. The definitive test is considered to be the more robust test given that increased replication was used and cell counts were performed daily giving more statistical power to the results analysis.

 

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

Description of key information

ErC50 (72h): >1.68 mg/L (Raphidocelis subcapitata, growth rate, based on the geometric mean measured concentration)

ErC10 (72h): 1.05 mg/L (Raphidocelis subcapitata, growth rate, based on the geometric mean measured concentration)

Key value for chemical safety assessment

Additional information

One experimental study was available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to the OECD 201 (GLP) using freshwater algae Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) (Goodband, 2020). The static test was carried out with the test item concentrations of control, 0.625, 1.25, 2.5, 5.0 and 10 mg/L (based on the nominal concentrations) and control, 0.549, 0.762, 1.15, 1.67 and 1.68 (based on the geometric mean measured concentration). At the start of the test (0 hours) the measured concentrations were shown to be 0.731, 1.05, 1.76, 2.74 and 2.68 mg/L (117, 84, 70, 55 and 27% of nominal, respectively). After 72 hours, the measured concentrations were shown to be 0.330, 0.464, 0.631, 0.990 and 0.877 mg/L (53, 37, 25, 20 and 9% of nominal, respectively).

Given that the analytical results showed a decline in measured concentration over the 72-hour test period, the results have been calculated based on geometric mean measured concentrations. The geometric mean measured concentrations were calculated to be 0.549, 0.762, 1.15, 1.67 and 1.68 mg/L (88, 61, 46, 33 and 17% of nominal, respectively). Based on geometric mean concentrations, the 72-hour EyC50 and ErC50 values were estimated to be 1.23 and >1.68 mg/L, respectively. The corresponding EC10 value for yield and growth rate were considered to be 0.645 and 1.05 mg/L. All validity criteria were met, therefore, the test was considered valid.