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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:
29 October - 18 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, 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:
(amended 2009)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Test concentrations were verified by chemical analysis. Water samples (3.0 mL) were taken from the control and all test concentrations at the start of the test and after 24, 48 and 72 hours.
At the intermediate sampling points, samples were taken from the additional replicates/concentration prepared for sampling purposes. At the end of the exposure period, samples were taken from one replicate of each test concentration.

Maintenance of actual concentrations was checked by running a test vessel at an intermediate test item concentration but without algae and samples for analysis were taken at the start, after 24, 48 and 72 hours.

Samples were stored in the freezer until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of test solutions started with a loading rate of 100 mg/L applying one day of magnetic stirring in a closed vessel in order to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one hour after which the clear and colourless Saturated Solution (SS) was siphoned out and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
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

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 test solution 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
Temperature was maintained between 22 and 23°C throughout the test.
pH:
t = 0: 7.3
t = 72h: 7.5 - 7.9
Nominal and measured concentrations:
Range-finding test:
Test concentrations: 0.1, 1.0, 10 and 100% of a saturated solution (SS) prepared at a loading rate of 100 mg/L.
Measured test concentrations (at test initiation): 0.51 and 5.3 mg/L at 10 and 100% of the saturated solution, respectively. Both concentrations decreased with time: to 30-32% of initial after 24 hours of exposure, to 7.6-18% of initial after 48 hours of exposure and finally below the limit of detection (LOD was 0.0127 mg/L) at the end of the test.

Final test:
Based on the results of the range-finding test the following dilutions of the SS prepared at a loading rate of 100 mg/L were assigned to the final test:4.6, 10, 22, 46 and 100%

Measured test concentrations are presented in Table 1 and Table 2 (in field 'Any other information on results').


Details on test conditions:
TEST SYSTEM
- Test vessel:
120 mL all-glass airtight capped vessels were used with no headspace, each containing 120 mL of test preparation for the control and each treatment group.
All test vessels were maintained under identical conditions.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth which was diluted to a cell density of 1 x 10^4 cells per mL prior to use.

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels without algae (replicates): 1-3 (at 22% of the SS)
- 2 extra replicates of each test concentration and the control for sampling purposes

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 (standard M2 medium was adjusted with a larger amount of NaHCO3 (300 mg/L), the addition of HEPES buffer (6 mmol/L) and a lower pH). The medium was prepared using reverse osmosis purified deionised water (Milli-RO, Millipore) and the pH adjusted to 7.1 ± 0.3.

- Illumination: continuously using TLD-lamps with a light intensity within the range of 87 - 90 µE.m-2.s-1 while constantly shaking.

EFFECT PARAMETERS MEASURED
- 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.
- Other: microscopic observations were performed on the control and the highest test concentration to observe for any abnormal appearance of the algae

OTHER
- Results used to determine the conditions for the definitive study:
The results of the range-finding test showed that the expected EC50 for inhibition of growth rate was >100% of the SS, and for inhibition of yield between concentrations obtained in 10 and 100% of an SS prepared at a loading rate of 100 mg/L. Based on this information the test concentrations of the definitive test were defined.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (December 2015)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.66 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 0.34 - 0.39
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.23 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: yes
- Observation of abnormalities: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: growth rate: 1.5 mg/L (95% CI: 1.4-1.6 mg/L)
- Other: >10% growth rate inhibition at and above 0.56 mg/L.

The 72h-ErC50 for the algal culture tested falls within the historical range at the test facility (0.82-2.3 mg/L).
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 or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on probit 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 measured concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Measured test substance concentrations (final test)

At the start of the test, the measured test concentrations were 0.22, 0.55, 1.0, 2.5, and 5.7 mg/L in 4.6, 10, 22, 46 and 100% of an SS prepared at a loading rate of 100 mg/L, respectively. All concentrations decreased during the test period. After 72 hours of exposure, the three lowest test concentrations were below the LOD, while the highest two concentrations were 0.16 and 0.53% of initial, respectively. The measured concentration in the sample taken from the 22% SS without algae also decreased but initially faster than in the vessel with algae but at the end of the test the remaining concentration in the vessel without algae was higher. Based on these results, the geometric mean measured concentrations were calculated.

Table 1: Concentrations of the test item in test medium - final test

Time of sampling
[hours]

Percentage of SS1
[%]

Analysed concentration
[mg/l]

Relative to
initial
[%]

 

 

 

 

0

0

n.d.

 

 

4.6

0.221

 

 

10

0.549

 

 

22

1.00

 

 

222

1.00

 

 

46

2.47

 

 

100

5.66

 

 

 

 

 

24

0

n.d.

n.a.

 

4.6

0.0828

37

 

10

0.288

52

 

22

0.414

42

 

222

0.261

26

 

46

1.32

53

 

100

2.34

41

 

 

 

 

48

0

n.d.

n.a.

 

4.6

0.0203

8.9

 

10

0.0273

4.9

 

22

0.0922

9.3

 

222

0.0183

1.8

 

46

0.233

9.4

 

100

0.476

8.4

 

 

 

 

72

0

n.d.

n.a.

 

4.6

n.d.

n.a.

 

10

n.d.

n.a.

 

22

n.d.

n.a.

 

222

0.0253

2.5

 

46

0.00403

0.16

 

100

0.0303

0.53

 

 

 

 

1          Percentage of a Saturated Solution (SS) prepared at a loading rate of 100 mg/l.

2          Without algae.

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

n.d.    Not detected. The limit of detection of the method was determined to be 0.0058 mg/L.

n.a.     Not applicable.

Table 2: Geometric mean measured concentrations

Pinyl Iso Butyraldehyde

%SS prepared at 100 mg/L

Measured concentration (mg/L)

Geomean measured conc. (mg/L)

t=0h

t=24h

t=48h

t=72 h

4.6

0.221

0.0828

0.020

0.00292

0.032

10

0.549

0.288

0.027

0.00292

0.059

22

1.00

0.414

0.0922

0.00292

0.10

221

1.00

0.261

0.018

0.025

0.10

46

2.47

1.32

0.233

0.0040

0.23

100

5.66

2.34

0.476

0.030

0.66

1Without algae

2Half the LOD

Inhibition results

Table 3: Percentage inhibition of growth rate during the final test

Geomean measured conc. of Pinyl Iso Butyraldehyde (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.784

0.0281

6

 

0.032

1.782

0.0177

3

0.1

0.059

1.768

0.0051

3

0.9

0.10

1.730

0.0248

3

3.1#

0.23

1.707

0.0151

3

4.3#

0.66

1.439

0.0140

3

19.4*

  * - effect was statistically significant

#- effect was statistically significant but biologically not relevant (<10%)

Microscopic observations

Microscopic observations at the end of the test in the highest test concentration revealed a normal and healthy appearance of the exposed cells when compared to the control.

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:
The ErC50, ErC10 and NOErC were >0.66, 0.37 and 0.23 mg/L.
Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata, strain: NIVA CHL 1. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, the algae were exposed to a Saturated Solution (SS) prepared at a loading rate of 100 mg/L and 4 dilutions of this SS (three replicate flasks per concentration) and a control (six replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature between 22 and 23°C. Preparation of test solutions started with a loading rate of 100 mg/L applying one day of magnetic stirring in a closed vessel in order to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one hour whereafter the clear and colourless Saturated Solution was siphoned out and used as highest test solution. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. Samples were taken from all treatments at t = 0 , 24, 48 and 72 h and analysed with a validated GC-FID method. All concentrations decreased during the test period. After 72 hours of exposure, the three lowest test concentration were below the LOQ, while the highest two concentrations were 0.16 and 0.53% of initial, respectively. Therefore the geometric mean measured concentrations were calculated and used for expression of endpoints. Statistically significant inhibition of growth rate was found at the three highest geometric mean measured concentration, starting at 0.10 mg/L. However, the effects at 0.10 and 0.23 mg/L, respectively, were biologically not relevant (<10%). Therefore the NOErC was set at 0.23 mg/L. The ErC10 and ErC50 based on geometric mean measured concentrations were 0.37 and >0.66 mg/L, respectively.

Description of key information

A study was performed to assess the effect of the test material on the growth of the green algae Pseudokirchneriella subcapitata, strain: NIVA CHL 1.The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, the algae were exposed to a Saturated Solution (SS) prepared at a loading rate of 100 mg/L and 4 dilutions of this SS (three replicate flasks per concentration) and a control (six replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature between 22 and 23°C. Preparation of test solutionsstarted with a loading rate of 100 mg/L applying one day of magnetic stirring in a closed vessel in order to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one hour whereafter the clear and colourless Saturated Solution was siphoned out and used as highest test solution. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium.Samples were taken from all treatments at t = 0 , 24, 48 and 72 h and analysed with a validated GC-FID method.All concentrations decreased during the test period. After 72 hours of exposure, the three lowest test concentration were below the LOQ, while the highest two concentrations were 0.16 and 0.53% of initial, respectively.Therefore the geometric mean measured concentrations were calculated and used for expression of endpoints. Statistically significant inhibition of growth rate was found at the three highest geometric mean measured concentration, starting at 0.10 mg/L. However, the effects at 0.10 and 0.23 mg/L, respectively, were biologically not relevant (<10%). Therefore the NOErC was set at 0.23 mg/L. The ErC10 and ErC50 based on geometric mean measured concentrations were 0.37 and >0.66 mg/L, respectively.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.37 mg/L

Additional information

EC50 growth rate is > 0.66 mg/l