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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
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control.

Frequency at t=0 h and t=72 h
Volume 4.8 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.

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 and at the end of the test period.

Additionally, reserve samples of 4.8 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:
no
Details on test solutions:
The batch of Dilithium tetraborate tested was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.

Combined limit/range-finding test: The test item was visually not completely soluble in test medium at the loading rate initially prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying a one-day period of magnetic stirring followed by a ten-minute period of ultrasonic waves to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by means of filtration through a 0.45 µm membrane filter (RC55, Whatman). pH of the obtained SS was set from 9.2 to 8.5 with 1 M HCl (Merck, Darmstadt, Germany). This solution was used as the highest 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.

Final test: after applying a one-day period of magnetic stirring followed by a ten-minute period of ultrasonic waves, the test item was observed to be completely dissolved in test medium Consequently, the filtration step was unnecessary. This solution was used as the highest concentration. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions 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 10(+4) cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
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.

- Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Reason for selection: This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

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 10(+4) cells/mL. The pre-culture was maintained under the same conditions as used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
Not stated
Test temperature:
21-24°C, constant within 2°C
pH:
6.0-9.0, preferably not varying by more than 1.5 unit
Dissolved oxygen:
Not stated.
Salinity:
Not stated.
Conductivity:
Not stated.
Nominal and measured concentrations:
Analytically confirmed nominal concentrations
Details on test conditions:
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1 x 10(+4) cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 83 to 86 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.
Replicates 3 replicates of each test concentration, 6 replicates of the control, 1 extra replicate of each test group for sampling purposes, 1 or 2 replicates of each test concentration without algae.
Medium: M2; 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

Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
36 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological relevance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical significance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Remarks on result:
other: based on biological relevance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Remarks on result:
other: based on statistical significance
Details on results:
Combined limit/Range-Finding Test; The mean cell densities were measured during the combined limit/range-finding test at 4 different concentrations of the test item and the control. No inhibition of neither growth rate nor yield was observed in the three lowest test concentrations. At the highest test concentration, 40 and 89% inhibition were observed for growth rate and yield, respectively. Therefore, the expected EC50 for growth rate inhibition was above the concentration obtained in the undiluted stock solution. The expected EC50 for yield inhibition was between 10 and 100% of the stock solution.

Final Test: A range of concentrations between 10 and 100% of the stock solution were tested to determine their effects on growth rate and yield. Growth rate and yield were in the range of the controls at the lowest test concentration during the 72 hour test period, whereas at higher concentrations growth rate and yield were increasingly inhibited. Statistically significant inhibition of growth rate and yield was found at test concentrations of 18 mg/L and higher. The inhibition of growth rate observed at the concentrations of 18 and 32 mg/L was however, considered biologically not relevant (i.e. were <10%). Therefore, the NOEC based on biological relevance was set at 32 mg/L. A biologically not relevant inhibition of yield was observed at the concertation of 18 mg/L, therefore, the NOEC based on biological relevance was set at 18 mg/L.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 56 mg/L when compared to the control.

Samples taken from all concentrations were analysed. Throughout the test duration, actual test concentrations were in agreement with nominal (91 – 103%). The concentrations measured in the samples without algae were comparable to the corresponding results of samples with algae.

Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations.

Table 1: Mean Cell Densities (x104Cells/mL) During the Combined Limit/Range-Finding Test

Time (h) 

Dilithium tetraborate; % SS prep. at 100 mg/L

Control

0.10

1.0

10

100

0

1.0

1.0

1.0

1.0

1.0

24

4.1

n.m.

n.m.

n.m.

3.3

48

40

n.m.

n.m.

n.m.

8.2

72

249

244

244

248

28

n.m. – not measured.

Table 2: Percentage Inhibition of Growth Rate During the Combined Limit/Range-Finding Test

Dilithium tetraborate

% SS prep. at 100 mg/L

Mean

Std. Dev.

n

%Inhibition

Control

1.838

0.0105

6

 

0.10

1.833

0.0079

3

0.3

1.0

1.832

0.0047

3

0.3

10

1.838

0.0045

3

0.0

100

1.105

0.0163

6

40

Table 3: Percentage Inhibition of Yield During the Combined Limit/Range-Finding Test

Dilithium tetraborate

% SS prep. at 100 mg/L

Mean

Std. Dev.

n

%Inhibition

Control

247.5

7.76

6

 

0.10

243.4

5.82

3

1.7

1.0

243.0

3.40

3

1.8

10

247.4

3.34

3

0.0

100

26.5

1.36

6

89

Table 4: Growth Rate and Percentage Inhibition for the Total Test period

Dilithium tetraborate

Nominal conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.915

0.0162

6

10

1.911

0.0119

3

0.2

18

1.882

0.0101

3

 1.7#

32

1.734

0.0276

3

 9.4#

56

1.414

0.0272

3

 26*

100

0.986

0.0166

3

 49*

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

Table 5: Growth Rate and Percentage Inhibition at Different Time Intervals

Dilithium tetraborate

Nominal conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.407

 

1.754

 

1.583

 

10

3

2.320

3.6

1.842

-5.0

1.572

0.7

18

3

2.165

10

1.840

-4.9

1.643

-3.8

32

3

1.938

20

1.619

7.7

1.645

-4.0

56

3

1.506

37

1.278

27

1.457

7.9

100

3

1.060

56

0.946

46

0.953

40

Table 6: Yield and Percentage Inhibition for the Total Test Period

Dilithium tetraborate

Nominal conc. (mg/L)

Mean

Std. Dev.

n

%Decrease

Control

311.6

14.73

6

 

10

308.3

10.91

3

1.1

18

282.6

8.50

3

 9.3#

32

181.0

14.95

3

 42*

56

68.6

5.57

3

 78*

100

18.3

0.96

3

 94*

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

Validity criteria fulfilled:
yes
Conclusions:
In an OECD TG 201 study with dilithium tetraborate, the EC50for growth rate inhibition (Pseudokirchneriella subcapitata) > 100 mg/L. The 72h-NOEC for growth rate inhibition was 32 mg/L based on biological relevance.
Executive summary:

The objective of the study (OECD TG 201) was to evaluate lithium tetraborate for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10 and EC50 for both inhibition of growth rate and inhibition of yield. The study was conducted according to OECD guideline No. 201.A final test was therefore conducted on growing algal cultures exposed to an untreated control and solutions containing 10, 18, 32, 56 and 100 mg/L of the test item. The initial algal cell density was 104cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and 72 hours of exposure.

 

Growth rate and yield were in the range of the controls at the lowest test concentration during the 72‑hour test period, whereas at higher concentrations growth rate and yield were increasingly inhibited. Statistically significant inhibition of growth rate and yield was found at test concentrations of 18 mg/L and higher. The inhibition of growth rate observed at the concentrations of 18 and 32 mg/L was however, considered biologically not relevant (i.e. were <10%). Therefore, the NOEC (inhibition of growth rate based on biological relevance) = 32 mg/L.

 

In conclusion, the EC50for growth rate inhibition (72h-ERC50) was beyond the range tested, i.e. exceeded the nominal concentration of 100 mg/L. The 72h-NOEC for growth rate inhibition was 32 mg/L based on biological relevance.

 

Description of key information

Growth rate and yield were in the range of the controls at the lowest test concentration during the 72‑hour test period, whereas at higher concentrations growth rate and yield were increasingly inhibited. Statistically significant inhibition of growth rate and yield was found at test concentrations of 18 mg/L and higher.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
32 mg/L

Additional information