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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 September 2017 - 27 October 2017
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 March 23, 2006; Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment
Version / remarks:
number 23, 2000
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h
Volume 2.0 mL
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Vehicle:
no
Details on test solutions:
The batch of the test substance tested was a colourless to light yellow viscous liquid with a purity of 97%. The test item was not completely soluble in test medium at the initial loading rate prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to accelerate and ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of 61-67 minutes. 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. Test solutions containing 32-100% of the SS were hazy at the end of the preparation procedure while lower test concentrations were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain: NIVA CHL 1
Source: In-house laboratory culture
-Method of cultivation: Algae stock cultures were started by inoculating growth medium 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. 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 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
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
Betwenn 22 and 23°C
pH:
7.9 - 8.3
Nominal and measured concentrations:
Nominal conc. (mg/L) Time weighted average (TWA) concentration (mg/L)
Control = Control
10 = 7.5
18 = 13
32 = 24
32 (without algae) = 23
56 = 39
100 = 69
Details on test conditions:
TEST SYSTEM:
- Test vessels: 100 mL, all-glass, containing 50 mL of test solution
- Medium: M2
- Cell density: An initial cell density of 1 x 104 cells/mL
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 91 to 93 µE/m2/s.
- 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

M2 medium; according to the OECD 201 Guideline, formulated using Milli-RO water and with 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 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

TEST CONCENTRATIONS
- The test substance: Solutions containing 10, 18, 32, 56 and 100% of a Saturated Solution (SS) prepared at a loading rate of 100 mg/L.
- Controls: Test medium without test item or other additives.
- Replicates:
3 replicates of each test concentration.
6 replicates of the blank control.
1 or 2 replicates of each test concentration without algae.
1 extra replicate of each test concentration for sampling purposes after 24 hours of exposure.

REFERENCE TEST
The objective of the present reference study was to evaluate Potassium dichromate for its ability to generate toxic effects in Pseudokirchneriella subcapitata (strain: NIVA CHL-1) during an exposure period of 72 hours and, if possible, to determine the EC50 for inhibition of both growth rate and yield (Test Facility No. 20134686).
Start of first exposure: 25 Sep 2017
Completion last exposure: 28 Sep 2017
The study procedures described in this report were based on the OECD guideline No. 201, Adopted March 23, 2006; Annex 5 corrected 28 July 2011 and ISO Standard 8692, Second edition, 01 October 2004.
This reference test was carried out to check the sensitivity of the test system used by Charles River Den Bosch to Potassium dichromate (Merck, Art. 1.04864, Batch K44879664).
Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 104 cells/ml.
The EC50 for growth rate inhibition (72h-ERC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 mg/L. 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.
The EC50 for yield inhibition (72h-EYC50) was 0.36 mg/L with a 95% confidence interval ranging from 0.35 to 0.36 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.
The study plan, raw data and report of this study are kept in the Charles River Den Bosch archives. The test described above was performed under GLP conditions with a QA-check.

Reference substance (positive control):
yes
Remarks:
K2Cr2O7 (potassium dichromoate). Algae were exposed for a period of 72 h to K2Cr2O7 concentration of 0.18; 0.32; 0.56; 1.0; 1.8; 3.2 mg/L. 72h-ERC50 and 72h-EYC50 lie in the historical ranges.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 13 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 68 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% confidence interval ranging from 66 to 70 mg/L.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 7.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 25 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
95% confidence interval ranging from 24 to 26 mg/L.

The effect parameters obtained in this study are summarized in the table below

Parameter (mg/L)

NOEC*

NOEC**

EC10

EC20

EC50

Growth rate

Value

<7.5

13

26

36

68

lower 95%-cl

 

 

24

35

66

upper 95%-cl

 

 

27

37

70

Yield

Value

<7.5

<7.5

8.3

12

25

lower 95%-cl

 

 

7.4

11

24

upper 95%-cl

 

 

9.2

13

26

Growth Rate And Percentage Inhibition For The Total Test Period

 

Test substance

TWA conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.882

0.0187

6

7.5

1.829

0.0077

3

2.84*#

13

1.803

0.0361

3

4.21*#

24

1.678

0.0399

3

10.83*

39

1.503

0.0231

3

20.14*

69

0.896

0.0293

3

52.37*

* effect was statistically significant;#effect biologically not relevant (<10%)

Yield And Percentage Inhibition For The Total Test Period

 

Test substance

TWA conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

282.6

16.21

6

7.5

240.3

5.55

3

14.95*

13

223.2

23.93

3

21.01*

24

153.3

18.02

3

45.73*

39

90.0

6.41

3

68.16*

69

13.8

1.32

3

95.13*

* effect was statistically significant

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, the test substance reduced growth rate and inhibited the yield of this fresh water algae species significantly at all of the TWA concentrations of the test substance tested.
The EC50 for growth rate inhibition (72h-ERC50) was 68 mg/L; the EC50 for yield inhibition (72h-EYC50) was 25 mg/L .
The 72h-NOEC for growth rate inhibition was 13 mg/L based on biological relevance; the 72h-NOEC for yield inhibition was below 7.5 mg/L
Executive summary:

The toxicity of the test substance to aquatic algae was assessed according to OECD guidelines and GLP principles. Normal exposure concentrations in the final tests were the following: 10, 18, 32, 56, 100 mg/L. The corresponding time weight average (TWA) concentrations were: 7.5, 13, 24, 39, 69 mg/L. It was decided to express effect concentration based on TWA concentrations since the test substance was not fully soluble in the test medium.

The test substance reduced the growth rate and inhibited the yield of this fresh water algae species significantly at all of the concentrations tested, under the conditions of the present study with Pseudokirchneriella subcapitata. A dose-related increase of inhibition of both growth rate and yield was observed at the end of the test resulting in an inhibition of 52% and 95% at the highest concentration tested for growth rate and yield, respectively.

The EC50 for growth rate inhibition (72h-ERC50) was 68 mg/L TWA; the EC50 for yield inhibition (72h-EYC50) was 25 mg/L TWA.

The 72h-NOEC for growth rate inhibition was 13 mg/L TWA based on biological relevance; the 72h-NOEC for yield inhibition was below 7.5 mg/L TWA.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Description of key information

The present study was performed according to OECD guideline No. 201, with Pseudokirchneriella subcapitata.

The test substance showed reduced growth rate and inhibited the yield of this fresh water algae species significantly.

The EC50 for growth rate inhibition (72h-ERC50) was 68 mg/L; the EC50 for yield inhibition (72h-EYC50) was 25 mg/L .

The 72h-NOEC for growth rate inhibition was 13 mg/L based on biological relevance; the 72h-NOEC for yield inhibition was below 7.5 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
68 mg/L
EC10 or NOEC for freshwater algae:
13 mg/L

Additional information

The toxicity of the test substance to aquatic algae and cyanobacteria was assessed according to OECD guidelines and GLP principles, using Pseudokirchneriella subcapitata

Exposure concentrations in the final tests : 0, 10, 18, 32, 56, 100 mg/L.

The corresponding time weight average (TWA) concentrations were: 7.5, 13, 24, 39, 69 mg/L. It was decided to express effect concentration based on TWA concentrations since the test substance was not fully soluble in the test medium.

The test substance reduced the growth rate and inhibited the yield of this fresh water algae species significantly at all of the concentrations tested.

Growth rate was inhibited for 2.84, 4.21, 10.83, 20.14 and 52.37%, at 7.5, 13, 24, 39 and 69 mg/L, respectively.

The EC50 for growth rate inhibition (72h-ERC50) was 68 mg/L TWA. The 72h-NOEC for growth rate inhibition was 13 mg/L TWA based on biological relevance; as the inhibition was below 10% at 7.5 and 13 mg/L. The study met the acceptability criteria prescribed by the study plan and was considered valid.