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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:
06 January 2017 - 20 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006 (Annex 5 corrected 28 July 2011)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008 (amended by EC No. 2016/266)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23
Version / remarks:
December 14, 2000
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and all test concentrations
- Sampling method: 6.0 mL, from the approximate centre of the test vessels, were taken for analysis from all test solutions at t=0 h, t=24 h, t=48h and t=72 h. At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling, instead samples were taken from replicate 1 of each group.
- Sample storage conditions before analysis: Samples were stored in a freezer (at <= -15°C) until analysis.
- Other: Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 and 48 hours of exposure and at the end of the test period.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: preparation of a saturated solution (direct addition) - In view of the substance being a multi-constituent constituted of two stereo-isomers, the test substance was treated as pure and therefore a Saturated Solution was used. Preparation of test solutions started with a loading rate of 100 mg/L applying a three- day period of magnetic stirring to accelerate dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 120 mL were added to each replicate vessel of the respective test concentration containing a 2.4 mL of an algal suspension providing a cell density of 10^4 cells/mL.
- Controls: Test medium without test item or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae (Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis)) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO water) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test.
- Culturing media and conditions (same as test or not): no: culture in M1, test in adjusted M2 medium
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/L (as CaCO3)
Test temperature:
22-23°C
pH:
t=0: 7.3
t=72 h: 7.4-7.8
Nominal and measured concentrations:
Nominal test concentrations (defintive test): 1.0, 3.2, 10, 32 and 100% of a saturated solution prepared at a loading rate of 100 mg/L
Corresponding geometric mean measured concentrations: 0.35, 1.2, 5.8, 21 and 66 mg/L

Measured concentrations in fresh solutions at test start were 77-92% of nominal. Measured concentrations in solutions at test end (t=72 h) were 10-59% of initial. See also field 'Any other information on results'.

Since test concentrations decreased gradually during the exposure period, effect concentrations were based on geometric mean measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 120 mL, all-glass, closed airtight with headspace reduced to a minimum
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 133.7 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of additional vessels: 1 or 3 replicates of each concentration without algae + 2 or 4 replicates of each group for sampling purposes on 24 and 48 hour of exposure

GROWTH MEDIUM
- Standard medium used: no (M1, according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water)
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap-water purified by reverse osmosis
- Culture medium different from test medium: yes (culture in M1, test in adjusted M2 medium (according to OECD 201, with 6x excess of NaHCO3 and addition of HEPES buffer (6 mmol/L))
- Intervals of water quality measurement: temperature - continuously in a separate vessel; pH - at the beginning and at the end of the test

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: not reported
- Photoperiod: continuous light
- Light intensity and quality: 84 to 87 µE.m-2.s-1 (from TLD-lamps)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.
- Other: At the end of the final test microscopic observations were performed in the control and the solution containing 10% SS to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3.2

RANGE FINDING STUDY
- Test concentrations: 1.0, 10 and 100% of a saturated solution prepared at a loading rate of 100 mg/L (measured concentration in the 100% saturated solution was ca. 86 mg/L at t=0 hours)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (February 2017)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
10.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% C.I. 9.0-12.3 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% C.I. 3.6-5.8 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no; Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 5.8 mg/L when compared to the control treatment.
- Aggregation of algal cells: no (not reported)
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no (not reported)
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72h-ErC50: 1.2 mg/L (95% C.I. 1.1-1.2 mg/L)
- Other: The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Data were tested for normality of distribution using Shapiro-Wilk´s Test on Normal Distribution: p(W) is smaller than or equal to the selected significance level of 0.010; therefore, treatment data significantly differ from normal distribution.
Data were tested for homogeneity of variance using Levene´s Test on Variance Homogeneity (with Residuals): The Levene test indicates variance heterogeneity (p <= 0.010).

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used.
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Step-down Jonckheere-Terpstra Test Procedure).
Eventually, Non-parametric Trend analysis by Contrasts (Monotonicity of Concentration/Response) revealed that linear trend is significant (p <= 0.05), and quadratic trend is not significant (p > 0.05). Therefore a user-selected multiple test was performed.

Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Samples taken from all groups were analyzed. The initially measured concentrations were at the level of 77-92% of nominal. Measured concentrations decreased gradually during the course of the test and were at the level of 10-59% of initial at the end of the exposure. Based on these results, the average exposure concentrations were calculated (geometric mean measured concentrations).

Final Test: Test Samples

Time of sampling
[hours]

Percentage of SS1

[%]

Analyzed concentration2
[mg/L]

Relative to
initial
[%]

0

0

n.d.

 

 

1.0

0.772

 

 

3.2

2.62

 

 

10

9.15

 

 

 103

8.95

 

 

32

27.6

 

 

100

88.5

 

 

 

 

 

24

0

n.d.

n.a.

 

1.0

0.592

77

 

3.2

2.14

82

 

10

7.13

78

 

 103

7.97

89

 

32

24.3

88

 

100

77.0

87

 

 

 

 

48

0

n.d.

n.a.

 

1.0

0.388

50

 

3.2

1.51

58

 

10

6.26

68

 

103

4.02

45

 

32

17.4

63

 

100

62.8

71

 

 

 

 

72

0

n.d.

n.a.

 

1.0

0.0879

11

 

3.2

0.253

10

 

10

2.68

29

 

 103

5.30

59

 

32

16.2

59

 

100

45.3

51

1      Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

2       Samples were stored in the freezer (≤ -15°C) until the day of analysis.

3         Without algae.

n.d.Not detected.

n.a.Not applicable.

Measured concentrations versus nominal concentrations

Citrolate

%SS prepared at 100 mg/L 

Measured concentration (mg/L)

Average exposure concentration (mg/L)

t=0h

t=24h

t=48h

t=72h

1.0

0.772

0.592

0.388

0.0879

0.35

3.2

2.62

2.14

1.51

0.253

1.2

10

9.15

7.13

6.26

2.68

5.8

10 WA

8.95

7.97

4.02

5.3

6.2

32

27.6

24.3

17.4

16.2

21

100

88.5

77

62.8

45.3

66

WA – without algae

Growth rates were in the range of the controls at the two lowest concentrations tested during the 72-hour test period, whereas the growth rate of algae exposed to 5.8 mg/L and higher, were increasingly reduced. Statistically significant inhibition of growth rate was found at the three highest concentrations.

Percentage inhibition of growth rate (total test period) during the final test

Citrolate

Average exposure conc. (mg/L) 

Mean

Std. Dev.

n

%Inhibition

Control

1.631

0.0233

6

0.35

1.557

0.0496

3

4.6

1.2

1.615

0.0237

3

1.0

5.8

1.354

0.0382

3

17.0*

21

0.219

0.3075

3

86.6*

66

0.000

0.0000

3

100.0*

* - effect was statistically significant

Percentage inhibition of growth rate at different time intervals during the final test

Citrolate

Average exposure conc. (mg/L) 

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.686

 

1.653

 

1.554

 

0.35

3

1.655

1.8

1.462

11.5

1.552

0.1

1.2

3

1.715

-1.7

1.527

7.6

1.604

-3.2

5.8

3

1.029

39.0

1.543

6.7

1.489

4.2

21

3

0.443

73.7

-0.436

126.4

0.649

58.2

66

3

0.000

100.0

0.000

100.0

0.000

100.0

Validity criteria fulfilled:
yes
Remarks:
see 'Overall remarks'
Conclusions:
The ErC50, ErC10 and NOEC for growth rate inhibition were 10.5, 4.7 and 1.2 mg/L, respectively.
Executive summary:

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. The test was performed under GLP.

Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring to accelerate dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. All lower test concentrations (1.0, 3.2, 10 and 32% (v/v) of the SS) were prepared by subsequent dilutions of the 100% SS in test medium. All final test solutions were clear and colourless.

The test was performed in closed airtight vessels with minimum headspace and with adjusted M2 medium. Pseudokirchneriella subcapitata was exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control). Samples were taken from all treatments at t = 0, 24, 48 and 72 h and analysed with a validated GC-FID method. Initially measured test concentrations ranged between 77-92% of nominal. Since test concentrations gradually decreased during the test period, geometric mean measured concentrations were calculated: 0.35, 1.2, 5.8, 21 and 66 mg/L, corresponding with 1.0%, 3.2%, 10%, 32% (v/v) and 100% of the SS prepared at a loading rate of 100 mg/L.

The ErC50, ErC10 and NOEC for growth rate inhibition were 10.5, 4.7 and 1.2 mg/L respectively, based on geometric mean measured concentrations.

Description of key information

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. The test was performed under GLP.

Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring to accelerate dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. All lower test concentrations (1.0, 3.2, 10 and 32% (v/v) of the SS) were prepared by subsequent dilutions of the 100% SS in test medium. All final test solutions were clear and colourless.

The test was performed in closed airtight vessels with minimum headspace and with adjusted M2 medium. Pseudokirchneriella subcapitata was exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control). Samples were taken from all treatments at t = 0, 24, 48 and 72 h and analysed with a validated GC-FID method. Initially measured test concentrations ranged between 77-92% of nominal. Since test concentrations gradually decreased during the test period, geometric mean measured concentrations were calculated: 0.35, 1.2, 5.8, 21 and 66 mg/L, corresponding with 1.0%, 3.2%, 10%, 32% (v/v) and 100% of the SS prepared at a loading rate of 100 mg/L.

The ErC50, ErC10 and NOEC for growth rate inhibition were 10.5, 4.7 and 1.2 mg/L respectively, based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
10.5 mg/L
EC10 or NOEC for freshwater algae:
4.7 mg/L

Additional information