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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:
16 April 2016 - 22 July 2016
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:
2006; Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008, amended in 2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and the control
- Sampling method:
Frequency: At t=0 h, t=24 h, t=48h and t=72 h
Volume: 4.0 mL
- Sample storage conditions before analysis: Not applicable, samples were analysed on the day of sampling

At the end of the exposure period, samples were taken from one of the replicates of each concentration.

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, 48 and 72 hours of exposure.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: water accommodated fraction (WAF)

The batch of Cedryl Acetate tested was a clear pale yellow to yellow liquid with a purity of 53.6% and the test item was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.

All test concentrations were prepared separately. Loading rates ranging from 0.1 to 100 mg/L were magnetically stirred in closed vessels for two days to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for 1-1½ hours whereafter the WAF was siphoned out through glass wool and used for testing.
The final test solutions were all clear and colourless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm) in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (adjusted M2) 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. The cell density was measured immediately before use.

After preparation, volumes of 120 mL were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. The total fill volume of each vessel was appr. 120 mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
23-24°C
pH:
t=0: 7.4
t=72 h: 7.4-7.7
Nominal and measured concentrations:
Combined limt/range-finding test - WAF loading rates: 1.0, 10 and 100 mg/L.

Final tests:
Based on the results of the range-finding test the following WAF loading rates were assigned to the final test: 0.10, 0.40, 1.6, 6.4, 26 and 100 mg/L.
Geometric mean measured test concentrations: 0.026, 0.037, 0.044, 0.41, 0.36 and 0.31 mg/L (see table 1 in field "any other information on results' for more details).

Geometric mean measured concentrations showed no dose-response curve, but since the measured concentrations at the end of the renewal period were not maintain within 80-120% of initial measured concentrations, effect parameters were based on geometric mean measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 120 mL all-glass, air-tight closed vessels with no headspace to prevent any loss of test item due to volatilisation; fill volume: 120 mL
The control group was maintained under identical conditions but not exposed to the test material.
- Initial cells density: 1 x 10^4 cells per mL
- No. of vessels per concentration (replicates): 3 + 2 extra replicates of each test concentration for sampling purposes
- No. of vessels per control (replicates): 6 +2 extra replicates of the control for sampling purposes
- No. of vessels without algae (replicates): 3 (of the WAF at loading rate of 1.6 mg/L)

GROWTH MEDIUM
- Stock culture medium: M1 prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture and test medium: adjusted M2 (prepared in accordance with OECD 201 using reverse osmosis purified deionised water (Milli-RO, Millipore); larger amount of NaHCO3, addition of HEPES buffer and a lower pH - pH adjusted to 7.1 ± 0.3)
- llumination: continuously using TLD-lamps with a light intensity within the range of 79 to 80 µE.m-2.s-1.
- Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Determination of cell concentrations: Cells were counted using a microscope and a counting chamber.
- Appearance of the cells: At the end of final test 2 microscopic observations were performed on all test concentrations and the control to observe for any abnormal appearance of the algae.

- Results used to determine the conditions for the definitive study:
The results of the combined limit/range-finding test showed that the expected EC50 for growth rate inhibition was above the concentration obtained in a WAF prepared at a loading rate of 100 mg/L. The expected EC50 for yield inhibition approximated the concentration obtained in a WAF at 10 mg/L.
In the combined limit/range-finding test, after 72 hours of exposure algal cells in all three WAFs were observed to clot. Therefore, algal cells were counted under a microscope during the final tests.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (July 2016)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.31 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on the fact that only 21% inhibition of growth rate was observed at this concentration
Duration:
72 h
Dose descriptor:
EC10
Basis for effect:
growth rate
Remarks on result:
not determinable
Duration:
72 h
Dose descriptor:
NOEC
Basis for effect:
growth rate
Remarks on result:
not determinable
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities in algal cells: normal and healthy appearance of the algal cells exposed to the three lowest concentrations (0.026-0.044 mg/L) when compared to the control. At the three highest concentrations (0.31-0.41 mg/L) algal cells were observed
to be deformed.

Statistically significant inhibition of both growth rate and yield was found at all test concentrations, i.e. at 0.026 mg/L and higher (see table in the field 'Any other information on results').

Growth rate inhibition at the highest tested rate (i.e. 0.31 mg/L, geometric mean measured concentration) was 21.2%, thus in absence of 50% effect a defined EC50 could not be derived.
No robust NOEC, EC10 and EC20-values could be determined for both growth rate and yield due to the fact that no dose-response curve could be obtained with the measured concentrations. In addition, no EC50 value for yield could be determined.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72h-ErC50: 1.0 mg/L (95% confidence interval ranging from 0.97 to 1.0 mg/L)
- Other: The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
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 and/or yield (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller).

Calculation of ECx values was based on weibit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield 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).

Combined limit/range-finding test

 

Percentage inhibition of growth rate during the combined limit/range-finding test

Cedryl Acetate, WAF at x mg/L

Mean

Std. Dev.

n

%Inhibition

Control

1.621

0.0250

6

1.0

1.486

0.0693

3

8.3

10

1.360

0.0064

3

16.1

100

1.174

0.0119

3

27.6

 

 

Measured test substance concentrations - final test 2

 

Analysis was performed on the day of sampling.

 

Table 1. Concentrations of the test item in test medium - final test 2

Time of sampling
[hours]

Loading rate1

[mg/L]

Concentration
analysed
[mg/L]

Relative to
initial
[%]

 

 

 

 

0

0

n.d.

 

 

0.1

0.0603

 

 

0.4

0.104

 

 

1.6

0.138

 

 

1.62

0.148

 

 

6.4

0.874

 

 

26

1.02

 

 

100

1.05

 

24

0

n.d.

 

 

0.1

<LOD

 

 

0.4

0.0523

50

 

1.6

0.0750

54

 

1.62

0.0993

67

 

6.4

0.712

81

 

26

0.682

67

 

100

0.500

48

48

0

n.d.

 

 

0.1

<LOD

 

 

0.4

<LOD

 

 

1.6

<LOD

 

 

1.62

<LOD

 

 

6.4

0.175

20

 

26

0.165

16

 

100

0.183

17

72

0

n.d.

 

 

0.1

<LOD

 

 

0.4

<LOD

 

 

0.44

<LOD

 

 

1.6

<LOD

 

 

1.62

<LOD

 

 

6.4

0.259

30

 

26

0.140

14

 

100

0.0924

9

 

 

 

 

1            A water accommodated fraction (WAF) prepared at the loading rate.

2            Without algae.

3            Estimated value, calculated by extrapolation of the calibration curve.

4            Vessel 2

<LOD  The limit of detection of the method (LOD) determined on 19 July 2016 was 0.0443 mg/L, on 20 July 2016 the LOD was 0.0260 mg/L and on 21 July 2016 the LOD was 0.0560 mg/L taking a dilution factor of 0.5 into account (i.e. a concentration factor of 2).

n.d.      Not detected.

n.a.      Not applicable.

 

Table 2. Measured concentrations versus WAFs prepared at various loading rates

 

Cedryl Acetate,

WAF at

x mg/L

Measured concentration (mg/L)

Geometric mean measured concentration (mg/L)

t=0h

t=24h

t=48h

t=72h

0.10

0.060

0.0222

0.0132

0.0282

0.026

0.40

0.104

0.052

0.0132

0.0282

0.037

1.6

0.138

0.0750

0.0132

0.0282

0.044

1.61

0.148

0.0993

0.0132

0.0282

0.048

6.4

0.874

0.712

0.175

0.259

0.41

26

1.02

0.682

0.165

0.140

0.36

100

1.05

0.500

0.183

0.0924

0.31

1Without algae

2Half the LOD

 

Inhibition results – final test 2

 

Table 3: Percentage inhibition of growth rate (total test period) during final test 2

Geometric mean measured conc. Cedryl Acetate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.530

0.0126

6

0.026

1.485

0.0104

3

2.9*

0.037

1.368

0.2102

3

10.6*

0.044

1.496

0.0244

3

2.2*

0.41

1.330

0.0388

3

13.1*

0.36

1.204

0.0388

3

21.3*

0.31

1.206

0.0298

3

21.2*

* - effect was statistically significant

 

Table 4: Percentage inhibition of yield during final test 2

 

Geometric mean measured conc. Cedryl Acetate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

97.5

3.70

6

0.026

85.1

2.67

3

12.8*

0.037

66.8

33.50

3

31.5*

0.044

88.1

6.37

3

9.7*

0.41

53.3

6.11

3

45.4*

0.36

36.3

4.27

3

62.8*

0.31

36.3

3.41

3

62.8*

* - effect was statistically significant

 

Validity criteria fulfilled:
yes
Remarks:
(In controls: cell density increased by an average factor of >16 within 72 hours, mean CV for section-by-section specific growth rates did not exceed 35% and CV of average specific growth rates during the whole test period did not exceed 7%)
Conclusions:
No robust NOEC and ECx-values could be determined for both growth rate and yield due to the fact that no dose-response curve could be obtained with the geometric measured concentrations. In addition, no EC50 value for yield could be determined. However, it can be safely stated that the EC50 for growth rate was >0.31 mg/L based on the fact that only 21% inhibition of growth rate was observed at this concentration.
Executive summary:

A study was performed to assess the effect of the test material on the growth of the green algaPseudokirchneriella subcapitata.The method followed that described in the OECD TG No 201. Water Accommodated Fractions (WAFs) were separately prepared in closed vessels at loading rates of 0.10, 0.40, 1.6, 6.4, 26 and 100 mg/L applying a 2-day period of magnetic stirring in closed vessels followed by a 1 -1.5 hour stabilisation period. The aqueous mixtures were siphoned out through glass wool and used as test solutions. The final test solutions were all clear and colourless.

Pseudokirchneriella subcapitatawas exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control) under constant illumination and shaking at a temperature of 23 - 24°C.

Samples were taken from all treatments at t = 0 , 24, 48 and 72 h and analysed with a validated GC-FID method.At the start of the test, the measured test concentrations were 0.060, 0.10, 0.14, 0.87, 1.0 and 1.1 mg/L in the WAFs prepared at loading rates of 0.10, 0.40, 1.6, 6.4, 26 and 100 mg/L, respectively. Theseconcentrations did not remain stable during the test period,the four lowest concentrations decreased below the limit of detection (LOD).Therefore the geometric mean measured concentrations were calculated.

The geometric mean measured concentrationsshowed no dose-response curve due to the fact that average exposure concentrations were similar at the three lowest WAFs, i.e. 0.026- 0.044 mg/L and at the three highest WAFs (0.31-0.41 mg/L). The mean measured concentrations at the start seem to reflect the exposure concentrations best. However, according to OECD Guideline 201 analysis of the results should be based on geometric mean concentrations during exposure in case the deviation from the measured initial concentration is not within the range of ± 20%. Effect parameters are therefore based on geometric mean measured concentrations.

Statistically significant inhibition of growth rate was found at all tested concentrations but in view of the lack of dose response either on geometric mean concentration or on measured concentrations at the start the determination of effect values cannot be reliably estimated. This means that no robust NOEC and ECx-values could be determined for both growth rate and yield. However, it can be safely stated that the EC50for growth rate was >0.31 mg/L based on the fact that only 21% inhibition of growth rate was observed at this concentration.

Description of key information

A study was performed to assess the effect of the test material on the growth of the green algaPseudokirchneriella subcapitata.The method followed that described in the OECD TG No 201. Water Accommodated Fractions (WAFs) were separately prepared in closed vessels at loading rates of 0.10, 0.40, 1.6, 6.4, 26 and 100 mg/L applying a 2-day period of magnetic stirring in closed vessels followed by a 1 -1.5 hour stabilisation period. The aqueous mixtures were siphoned out through glass wool and used as test solutions. The final test solutions were all clear and colourless.

Pseudokirchneriella subcapitatawas exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control) under constant illumination and shaking at a temperature of 23 - 24°C.

Samples were taken from all treatments at t = 0 , 24, 48 and 72 h and analysed with a validated GC-FID method.At the start of the test, the measured test concentrations were 0.060, 0.10, 0.14, 0.87, 1.0 and 1.1 mg/L in the WAFs prepared at loading rates of 0.10, 0.40, 1.6, 6.4, 26 and 100 mg/L, respectively. Theseconcentrations did not remain stable during the test period,the four lowest concentrations decreased below the limit of detection (LOD).Therefore the geometric mean measured concentrations were calculated.

The geometric mean measured concentrationsshowed no dose-response curve due to the fact that average exposure concentrations were similar at the three lowest WAFs, i.e. 0.026- 0.044 mg/L and at the three highest WAFs (0.31-0.41 mg/L). The mean measured concentrations at the start seem to reflect the exposure concentrations best. However, according to OECD Guideline 201 analysis of the results should be based on geometric mean concentrations during exposure in case the deviation from the measured initial concentration is not within the range of ± 20%. Effect parameters are therefore based on geometric mean measured concentrations.

Statistically significant inhibition of growth rate was found at all tested concentrations but in view of the lack of dose response either on geometric mean concentration or on measured concentrations at the start the determination of effect values cannot be reliably estimated. This means that no robust NOEC and ECx-values could be determined for both growth rate and yield. However, it can be safely stated that the EC50for growth rate was >0.31 mg/L based on the fact that only 21% inhibition of growth rate was observed at this concentration.

Key value for chemical safety assessment

Additional information

No robust NOEC and ECx-values could be determined for growth rate. The ErC50 is >0.31 mg/L based on the fact that only 21% inhibition of growth rate was observed at this concentration.