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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:
15 May 2016 - 14 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
07 December 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Version / remarks:
December 14, 2000
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken from all test concentrations and the control at t=0 h, t=24 h, t=48h and t=72 h.
Vehicle:
no
Details on test solutions:
The test item was not completely soluble in test medium at the loading rates initially prepared. All test solutions were prepared separately and started with loading rates ranging from 0.32 to 100 mg/L applying two days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for approximately 1¼ hours where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used as the test concentrations.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
The test was carried out using an in-house laboratory culture of Pseudokirchneriella subcapitata, strain: NIVA CHL 1. Algae stock cultures were started by inoculating growth medium (M1 medium) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s) in a climate room at a temperature of 21-24°C.

PRE-CULTURE
3 days before the start of the test, cells from the algal stock culture were inoculated in M2 culture medium (same medium used for the range-finding and definitive tests) at a cell density of 10000 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Temperature of medium was measured continuously in a temperature control vessel and was maintained at 22-23°C throughout the test.
pH:
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.3 - 7.7.
Nominal and measured concentrations:
Nominal concentrations: WAFs at 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.

Analysis of the samples taken at the start of the test showed measured concentrations of 0.17, 0.40, 1.4, 4.3, 22 and 18 mg/L in the WAFs at 0.32, 1.0, 3.2, 10, 32 and 100 mg/L, respectively. All, except the highest test concentration decreased during the test period. The measured concentration in the sample taken from the WAF at 3.2 mg/L without algae also decreased but less than in the vessel with algae. Given this result, the EC50 values were expressed in terms of the average (geometric mean) exposure concentrations. See Table below.

Geraldehyde,
WAF at
x mg/L Measured concentration (mg/L) Average (geometric mean) exposure concentration (mg/L)
t=0h t=24h t=48h t=72h
0.32 0.173 0.125 0.054 0.0036 0.045
1.0 0.397 0.214 0.125 0.016 0.11
3.2 1.41 1.18 0.381 0.186 0.59
3.2(A) 1.43 1.27 1.21 0.859 1.2
10 4.33 4.28 2.65 1.38 2.9
32 22 20.0 15.4 9.00 16
100 18.0 21.6 15.6 18.6 18
(A) Without algae
Details on test conditions:

TEST SYSTEM
- Test vessel:
120 mL, airtight closed vessels with no headspace to prevent any loss of the test item due to volatilization were used for the control and each treatment group.
The control group was maintained under identical conditions but not exposed to the test material.

- Initial cells density:
After preparation of the test solutions, volumes of 120 mL were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension (pre-culture) was added to each replicate providing a cell density of 10000 cells/mL.

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None (not applicable)

GROWTH MEDIUM
- Standard medium used: no
Adjusted M2; Standard M2 according to the OECD 201 Guideline but with a larger amount of NaHCO3, the addition of HEPES buffer and a lower pH . This medium will be formulated using Milli-RO water and will have the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 300 mg/L
HEPES buffer 6 mmol/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 7.1 ± 0.3

- Photoperiod:
Continuousl light using TLD-lamps with a light intensity within the range of 87 to 91 µE.m-2.s-1. Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

- Salinity:
Not applicable

- Determination of cell concentrations: spectrophotometer
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a spectrophotometer. Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

- Results used to determine the conditions for the definitive study:
A range finding study was conducted at nominal concentrations of 0 (control), 1.0, 10 and 100 mg/L. At 1.0, 10 and 100 mg/L, respectively, the percent inhibition of growth rate was 2.5, 26.9 and 58.2%, and the percent inhibition of yield was 14.7, 78.7 and 96.7%. Based on this information test concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (tested between 18 and 21 July 2016)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.9 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.:
0.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.045 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Please refer to the tables under "Any other information on results incl. tables".
Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ERC50) was 1.0 mg/L with a 95% confidence interval ranging from 0.97 to 1.0 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.43 mg/L with a 95% confidence interval ranging from 0.42 to 0.44 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range
Reported statistics and error estimates:
The p(Chi2) value for the overall fit for growth rate was 1.00000.
The p(F) value for the slope of the regression curve for growth rate was <0.001.
For confidence intervals of EC-values please refer to the table with effect parameters under "Any other information on results incl. tables".

Detailed results on percentage inhibition of growth rate and all effect parameters for growth rate are presented in the table below.

Table 1: Percentage inhibition of growth rate (total test period) during the final test

Average conc. Geraldehyde (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.631

0.0862

6

0.045

1.596

0.0255

3

2.2

0.11

1.518

0.0256

3

6.9*

0.59

1.345

0.0358

3

17.5*

2.9

0.745

0.2280

3

54.3*

16

0.275

0.0165

3

83.1*

18

0.408

0.0798

3

75.0*

* Effect was statistically significant

Effect parameters for growth rate

Parameter (mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Value

0.045

0.21

0.52

2.9

lower 95%-cl

 

0.14

0.38

2.5

upper 95%-cl

 

0.30

0.68

3.5

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

Time of sampling
[hours]

Loading rate1

[mg/L]

Concentration
analysed
[mg/L]

Relative to
initial
[%]

 

 

 

 

0

0

n.d.

 

 

0.32

0.173

 

 

1.0

0.397

 

 

3.2

1.41

 

 

3.22

1.43

 

 

10

4.33

 

 

32

223, 4

 

 

100

18.0

 

 

 

 

 

24

0

n.d.

n.a.

 

0.32

0.125

72

 

1.0

0.214

54

 

3.2

1.18

84

 

3.22

1.27

89

 

10

4.28

99

 

32

20.03

90

 

100

21.6

120

 

 

 

 

48

0

n.d.

n.a.

 

0.32

0.0544

31

 

1.0

0.125

31

 

3.2

0.381

27

 

3.22

1.21

85

 

10

2.65

61

 

32

15.43

69

 

100

15.6

86

 

 

 

 

72

0

n.d.

n.a.

 

0.32

n.d.

n.a.

 

1.0

0.0164

4.0

 

3.2

0.186

13

 

3.22

0.859

60

 

10

1.38

32

 

32

9.00

40

 

100

18.6

103

 

 

 

 

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

2  Without algae.

3  Samples were re-diluted on 14-Jul-2016 and analysed, because the first results were extrapolated.

4  Estimated value, calculated by extrapolation of the calibration curve. LOD is 0.0071 mg/L.

n.d.    Not detected.

n.a.     Not applicable.

Table 3 WAF’s, measured and average exposure concentrations

Geraldehyde, WAF at

x mg/L

Measured concentration (mg/L)

Average exposure concentration (mg/L)

t=0h

t=24h

t=48h

t=72h

0.32

0.173

0.125

0.054

0.0036

0.045

1.0

0.397

0.214

0.125

0.016

0.11

3.2

1.41

1.18

0.381

0.186

0.59

3.21

1.43

1.27

1.21

0.859

1.2

10

4.33

4.28

2.65

1.38

2.9

32

22

20.0

15.4

9.00

16

100

18.0

21.6

15.6

18.6

18

1Without algae

2Half the LOD

Validity criteria fulfilled:
yes
Remarks:
In the control, cell density increased by an average factor of at least 16 (i.e. 137). The mean CV for section-by-section specific growth rates was <35% (i.e. 11%). The CV of average specific growth rates during the whole test period was <7% (i.e. 5%).
Conclusions:
The ErC50, ErC10 and NOEC were 2.9, 0.21 and 0.045 mg/L, respectively.
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. Following a preliminary range-finding test, the algae were exposed to solutions of the test material at nominal concentrations of0.32, 1.0, 3.2, 10, 32 and 100mg/L (three replicate flasks per concentration, 6 replicates for the control) for 72 hours, under constant illumination and shaking at a temperature of 22 -23 °C.

The test item was not completely soluble in test medium at the loading rates initially prepared. All test solutions were prepared separately and started with loading rates ranging from 0.32 to 100 mg/L applying two days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for approximately 1¼hours where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used as the test concentrations.

The initial algal cell density was 104cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of exposure concentrations were taken at the start, after 24, 48 and 72 hours of exposure.

Analysis of the samples taken at the start of the test showed measured concentrations of 0.17, 0.40, 1.4, 4.3, 22 and 18 mg/L in the WAFs at 0.32, 1.0, 3.2, 10, 32 and 100 mg/L, respectively. All, except the highest test concentration decreased during the test period. The measured concentration in the sample taken from the WAF at 3.2 mg/L without algae also decreased but less than in the vessel with algae. Given this result, the EC50values were expressed in terms of the average exposure concentrations that were calculated to be: 0.045, 0.11, 0.59, 1.2, 2.9, 16 and 18 mg/L.

The study met the acceptability criteria. The ErC50 is 2.9, the ErC10 is 0.21 and the NOEC is 0.045 mg/l.

Description of key information

The EC50 for Geraldehyde to green alga (OECD TG 201): 2.9 mg/L

The EC10 for Geraldehyde (OECD TG 201): 0.21 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
2.9 mg/L
EC10, LC10 or NOEC for freshwater algae:
0.21 mg/L

Additional 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. Following a preliminary range-finding test, the algae were exposed to solutions of the test material at nominal concentrations of0.32, 1.0, 3.2, 10, 32 and 100mg/L (three replicate flasks per concentration, 6 replicates for the control) for 72 hours, under constant illumination and shaking at a temperature of 22 -23 °C.

The test item was not completely soluble in test medium at the loading rates initially prepared. All test solutions were prepared separately and started with loading rates ranging from 0.32 to 100 mg/L applying two days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for approximately 1¼hours where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used as the test concentrations.

The initial algal cell density was 104cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of exposure concentrations were taken at the start, after 24, 48 and 72 hours of exposure.

Analysis of the samples taken at the start of the test showed measured concentrations of 0.17, 0.40, 1.4, 4.3, 22 and 18 mg/L in the WAFs at 0.32, 1.0, 3.2, 10, 32 and 100 mg/L, respectively. All, except the highest test concentration decreased during the test period. The measured concentration in the sample taken from the WAF at 3.2 mg/L without algae also decreased but less than in the vessel with algae. Given this result, the EC50values were expressed in terms of the average exposure concentrations that were calculated to be: 0.045, 0.11, 0.59, 1.2, 2.9, 16 and 18 mg/L.

The study met the acceptability criteria. The ErC50 is 2.9, the ErC10 is 0.21 and the NOEC is 0.045 mg/l.