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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:
Study initiation date : 31/10/2013, study completion date : 12/05/2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentrations of the test substance were determined for the replicates with alga inoculums at the beginning and the end of the test. Before sampling, separation of algae from the test medium was made by syringe filters. For the extra replicates, every 24 hours after the test initiation, samples were taken for chemical analysis.
Vehicle:
no
Details on test solutions:
Range-finding test :
During the pre-test , a white precipitation was observed in the test solution of the treatment groups. The test substance (molecular formula: Li3AlF6) contains aluminum (Al) element, so the precipitation could result from the formation of low soluble salts, i.e., the reaction product between aluminum and anions present in the test water. The test solution of each treatment was prepared at its respective nominal concentration: a certain amount of the test substance was added into the alga medium, and stirred (via magnetic stirrer) for 72h at 500rpm in the dark followed by filtration using a 0.45μm nitrocellulose membrane. After filtration the test solutions were clear and no precipitation was visible.

Definitive test :
The test solution of each treatment was prepared at its respective nominal concentration: a certain amount of the test substance was added into the alga medium, and stirred (via magnetic stirrer) for 72h at 500rpm in the dark followed by filtration using a 0.45μm nitrocellulose membrane. After filtration the test solutions were clear and no precipitation was visible.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was performed with Pseudokichneriella subcapitata (ID: PS-IHB-130822) which was originally supplied by the institute of hydrobiology, Chinese Academy of Sciences. This common species is one of the species recommended in the OECD TG 201. Ease of culturing and handling, sensitivity to a variety of chemical substances, and the extensive data base for this species make them suitable for acute toxicity testing.

For evaluation of the algal quality and experimental conditions, potassium dichromate was tested as a positive control twice in 2013 to demonstrate satisfactory test conditions. The 72h-ErC50 values of the test substance to Pseudokirchneriella subcapitata were 0.83mg/L (95% CI 0.76-0.89mg/L) and 0.84mg/L (95% CI 0.74-0.92mg/L) , respectively, which were within the defined range of 1.10±0.48mg/L, recommended based on an international ring test(Pattard et al, 2009).

The algae were inoculated in 250mL glass flasks with a concentration of 10E4 cells/mL, incubated during 3 days and then inoculated to another flask. At the beginning of the test, the algae were in the exponential growth phase when inoculated into the test solutions.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
Measured from 22.9 to 23.5°C in the definitive study.
pH:
Measured from 8.15 to 9.52 in the blank control of the definitive study.
Measured from 6.73 to 8.07 in the treatment groups of the definitive study.
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Range-finding test: three nominal concentrations of 12.0mg/L, 60.0mg/L and 120mg/L were used. The concentrations were not measured.
Definitive study: five nominal concentrations of 3.00mg/L, 5.40mg/L, 9.60mg/L, 16.8mg/L and 30.0mg/L respectively corresponding to geometric mean measured concentrations of fluoride: 2.14mg/L, 3.66mg/L, 6.20mg/L, 10.4mg/L and 17.9mg/L and geometric mean measured concentrations of aluminum: 0.0437mg/L, 0.121mg/L, 0.148mg/L, 0.476mg/L and 1.58mg/L.
Details on test conditions:
Algal medium : Analytical grade salts were dissolved in deionised water to obtain separate alga medium stock solutions. The stock solutions were filtrated with 0.22μm filter membrane and kept at 4°C. The alga medium was prepared by diluting the stock solutions in deionised water shortly before the test began.

Test vessels: 250 mL glass conical flasks

Conditions of exposure:
- Incubation: Each flask was capped with Parafilm and put in a culturing chamber by shaking at a speed of 130rpm.
- Duration: 72h;
- Temperature: 21-24℃;
- Illumination: the surface where the cultures are incubated should receive continuous, uniform fluorescent illumination at equivalent range of 4440–8880 lux for cool white light.

Procedure:
Test solutions: Before the initiation of the test, pH values of the respective test solution were adjusted to approximately 7.0 by 1M HCl. Three replicates were included for each treatment as well as six replicates for the blank control . Because of relatively large sampling volumes , one additional replicate with test solution was prepared for each concentration level for chemical analysis during the test. These replicates were treated identically to those used for testing but no alga inoculum was added.

Inoculation and incubation: An inoculum culture in the test medium was pre-cultured for 3 days before the start of the test under the test conditions. At the beginning of the test, the algae were in the exponential growth phase when inoculated to the test solutions and the density of the alga was 1.730×10E6/mL and 1.945x10E6/mL in the range-finding study and the definitive study respectively. In the range-finding study 1.73 mL of the alga culture and in the definitive study 1.54 mg/L of the alga culture were inoculated into 300mL of test solution for each treatment to obtain an initial cell density of the test solutions of about 10E4 /mL. 100mL of test solution was added into each of 250mL glass conical flasks.

Obervations and measurements: The density of alga cells in each flask was counted and light density in the culturing chamber was determined at 0, 24, 48 and 72 hours.At the beginning of the test, pH values for the control and each treatment were measured using the remaining test solutions. At the end of the test, one vessel was randomly taken from the treatment group and the control group for pH measurement and microscopic observation of the algae. The concentrations of the test substance were determined at the beginning and the end of the test. Before sampling, separation of algae from the test medium was made by syringe filters.

Results of the range-finidng study to define the concentrations in the definitive study:
Mean specific growth rate inhibition at 72h :
40.9% at 12 mg/L
92.9% at 60 mg/L
91.6% at 120 mg/L.

Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 11.3 - 23.8 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 CI : 3.95 - 11.4 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.57 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI : 1.46 - 11 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.44 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI : 0.177 - 4.17 mg/L
Details on results:
- Analytical measurements of the test item:
In the definitive test, the measured concentrations of fluoride for all treatment groups varied within ± 20% from their initial ones (-7.00% -14.5%) and the measured concentrations of aluminum also varied within ± 20% (-9.06% -15.1%) except the exposure concentration at 5.40 mg/L.
Since the test substance is a metal compound consisting of several elements (Li, Al and F), the results were calculated using the nominal concentrations in order to reflect the toxicity of the test substance.

- Validity criteria:
- Exponential growth in the control : yes. The mean biomass increase in the control cultures was 116 , which is higher than the validity criterion of at least 16 within the 72-hour test period.
The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 24.9%, which was below 35%.
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.43% which was below 7%.
The test fulfilled the above requirements and was therefore considered to be valid.

At the end of the test, no abnormal alga cells were observed in the control group by microscopic examination, but swollen cells were seen at the concentration of 30.0mg/L.


Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate was tested as a positive control twice in 2013 to demonstrate satisfactory test conditions. The 72h-ErC50 values of the test substance to Pseudokirchneriella subcapitata were 0.83mg/L (95% CI 0.76-0.89mg/L) and 0.84mg/L (95% CI 0.74-0.92mg/L) , respectively, which were within the defined range of 1.10±0.48mg/L, recommended based on an international ring test(Pattard et al, 2009).

Table 1: The measured concentrations offluoride inthe testsolutionsin the definitive test

Group

Measured Concentration (mg/L)

Geometric mean (mg/L)

72h vs.0h (%)

0h

72h

Blank Control

ND2

ND2

Treatment Group

(Nominal concentration,mg/L)

3.00

2.00

2.29

2.14

14.5

5.40

3.59

3.74

3.66

4.18

9.60

6.21

6.20

6.20

-0.16

16.8

10.4

10.5

10.4

0.96

30.0

18.6

17.3

17.9

-7.00

LOD2=0.0665mg/L;ND2, not detected;“—”: not applicable

Table 2: The measured concentrations ofalumimun inthe testsolutionsin the definitive test

Group

Measured Concentration (mg/L)

Geometric mean (mg/L)

72h vs.0h (%)

0h

72h

Blank Control

0.00177

0.00543

Treatment Group

(Nominal concentration,mg/L)

3.00

0.0408

0.0468

0.0437

14.7

5.40

0.0974

0.151

0.121

54.8

9.60

0.138

0.159

0.148

15.1

16.8

0.496

0.457

0.476

-7.87

30.0

1.66

1.51

1.58

-9.06

“—”: not applicable

 

Table 3: Cell density ofPseudokirchneriella subcapitataexposed to the test substance for 72 hours in the definitive test

Group

Cell density (cell/mL)

0h

24h

48h

72h

Blank Control

1

10000

69360

302200

1014000

2

10000

71220

410600

1316000

3

10000

73730

362200

1207000

4

10000

71220

326900

1019000

5

10000

74300

291300

1101000

6

10000

76200

374000

1290000

Mean

10000

72672

344533

1157833

CV (%)

0

3.45

13.3

11.5

Treatment Group

(Nominal concentration,mg/L)

3.00

1

10000

73330

337000

894800

2

10000

68560

304400

844700

3

10000

72760

378900

946300

Mean

10000

71550

340100

895267

CV (%)

0

3.64

11.0

5.67

5.40

1

10000

76500

285300

795300

2

10000

64560

211400

776600

3

10000

71970

345100

794100

Mean

10000

71010

280600

788667

CV (%)

0

8.5

23.9

1.33

9.60

1

10000

68490

235900

436400

2

10000

71040

269100

428600

3

10000

71700

274000

446400

Mean

10000

70410

259667

437133

CV (%)

0

2.41

7.98

2.04

16.8

1

10000

40460

113900

151000

2

10000

38520

78180

95810

3

10000

27858

80250

116800

Mean

10000

35613

90777

121203

CV (%)

0

19.1

22.1

23.0

30.0

1

10000

11630

13660

14940

2

10000

11770

12310

16700

3

10000

11850

13800

15780

Mean

10000

11750

13257

15807

CV (%)

0

0.95

6.21

5.57

 

Table 4: Growth of Pseudokirchneriella subcapitata for the blank control cultures in the definitive test

Replicate

Average specific growth rate

Yield

0-24h

24-48h

48-72h

CV (%) for

Section-by-section

0-72h

1

0.0807

0.0613

0.0504

23.8

0.0642

1004000

2

0.0818

0.0730

0.0485

25.4

0.0678

1306000

3

0.0832

0.0663

0.0502

24.8

0.0666

1197000

4

0.0818

0.0635

0.0474

26.8

0.0642

1009000

5

0.0836

0.0569

0.0554

24.2

0.0653

1091000

6

0.0846

0.0663

0.0516

24.4

0.0675

1280000

Mean

0.0826

0.0646

0.0506

24.9

0.0659

1147833

CV (%) for replicates

1.72

7.47

7.91

-

2.43

11.6

“—”: not applicable

 

Table 5: Growth ofPseudokirchneriella subcapitataexposed to the test substance in the definitive test

Treatment Group

(Nominal concentration,mg/L)

Average specific growth rate

Yield

0-24h

24-48h

48-72h

0-72h

72h-

Inhibition (%)

0-72h

72h-

Inhibition (%)

3.00

1

0.0830

0.0635

0.0407

0.0624

5.31

884800

22.9

2

0.0802

0.0621

0.0425

0.0616

6.53

834700

27.3

3

0.0827

0.0688

0.0381

0.0632

4.10

936300

18.4

Mean

0.0820

0.0648

0.0404

0.0624

5.31

885267

22.9

CV (%)

1.87

5.45

5.48

1.28

5.74

5.40

1

0.0848

0.0548

0.0427

0.0608

7.74

785300

31.6

2

0.0777

0.0494

0.0542

0.0604

8.35

766600

33.2

3

0.0822

0.0653

0.0347

0.0608

7.74

784100

31.7

Mean

0.0816

0.0565

0.0439

0.0607

7.94

778667

32.2

CV (%)

4.40

14.3

22.3

0.38

1.34

9.60

1

0.0802

0.0515

0.0256

0.0524

20.5

426400

62.9

2

0.0817

0.0555

0.0194

0.0522

20.8

418600

63.5

3

0.0821

0.0559

0.0203

0.0528

19.9

436400

62.0

Mean

0.0813

0.0543

0.0218

0.0525

20.4

427133

62.8

CV (%)

1.23

4.48

15.37

0.58

2.09

16.8

1

0.0582

0.0515

0.0256

0.0377

42.8

141000

87.7

2

0.0562

0.0555

0.0194

0.0314

52.4

85810

92.5

3

0.0821

0.0559

0.0203

0.0341

48.3

106800

90.7

Mean

0.0655

0.0543

0.0218

0.0344

47.8

111203

90.3

CV (%)

22.00

4.5

15.4

9.19

25.1

30.0

1

0.0063

0.0067

0.0037

0.0056

91.5

4940

99.6

2

0.0068

0.0019

0.0127

0.0071

89.2

6700

99.4

3

0.0071

0.0063

0.0056

0.0063

90.4

5780

99.5

Mean

0.0067

0.0050

0.0073

0.0063

90.4

5807

99.5

CV (%)

6.03

53.3

65.0

11.9

15.2

“—”: not applicable

 

 

Validity criteria fulfilled:
yes
Remarks:
OECD TG201 validity criteria fulfilled, see section "details on results"
Conclusions:
The toxicity results of the test substance to Pseudokichneriella subcapitata were the following: 72hErC50 = 16.1 mg/L and 72hErC10 = 7.57 mg/L.
Executive summary:

The growth inhibition effect of Lithium cryolite to Pseudokirchneriella subcapitata was determined in a 72 hours test according to the OECD TG 201 and under GLP.

During the pre-test, a white precipitation was observed in the test solution of the treatment groups. The test substance (molecular formula: Li3AlF6) contains aluminum (Al) element, so the precipitation could result from the formation of low soluble salts, i.e., the reaction product between aluminum and anions present in the test water. The test solution of each treatment was prepared at its respective nominal concentration: a certain amount of the test substance was added into the alga medium, and stirred (via magnetic stirrer) for 72h at 500 rpm in the dark followed by filtration using a 0.45μm nitrocellulose membrane.

Based on the results of the range-finding test, five nominal concentrations of 3.00 mg/L, 5.40 mg/L, 9.60 mg/L, 16.8 mg/L and 30.0mg/L (geometric mean measured concentrations of fluoride: 2.14 mg/L, 3.66 mg/L, 6.20 mg/L, 10.4 mg/L and 17.9 mg/L; those of aluminum: 0.0437 mg/L, 0.121 mg/L, 0.148 mg/L, 0.476 mg/L and 1.58 mg/L) were used in the definitive test. One control group of alga medium was run in addition to the treatment groups. Three replicates were included at each concentration as well as six replicates for the blank control .The test duration was 72h.

The mean biomass increase in the control cultures was 116, which is higher than the validity criterion of at least 16 within the 72-hour test period. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 24.9%, which was below 35%. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.43%, which was below 7%. Hence, the test was considered to be valid.

The measured concentrations of fluoride (F) analysed by ion chromatography (IC) for all treatment groups varied within ±20% from their initial concentrations (-7.00%-14.5%). The measured concentration of aluminum (Al) analysed by ICP-MS also varied within ±20% (-9.06%-15.1%) except the exposure concentration at 5.40 mg/L.

Since the test substance is a metal compound consisting of several elements (Li, Al and F), the results were calculated using the nominal concentrations in order to reflect the toxicity of the test substance. Based on average specific growth rate, the 72h-ErC10 value of the test substance to Pseudokirchneriella subcapitata was 7.57 mg/L (95% confidence limits: 1.46 mg/L–11.0 mg/L), and the 72h-ErC50 was 16.1 mg/L (11.3 mg/L–23.8 mg/L) . Based on yield, the 72h-EyC10 was 2.44 mg/L (0.177 mg/L– 4.17 mg/L), and the 72h-EyC50 was 6.96 mg/L (3.95 mg/L–11.4 mg/L).

Description of key information

A reliable toxicity test is available.

Based on the growth rate inhibition, the 72h ErC50 was determined to be 16.1 mg/L and the 72h ErC10 was determined to be 7.57 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
16.1 mg/L
EC10 or NOEC for freshwater algae:
7.57 mg/L

Additional information

One reliable key study is available for this endpoint. In this study, the growth inhibition effect of Lithium cryolite to Pseudokirchneriella subcapitata was determined in a 72 hours test according to the OECD TG 201 and under GLP.

During the pre-test, a white precipitation was observed in the test solution of the treatment groups. The test substance (molecular formula: Li3AlF6) contains aluminum (Al) element, so the precipitation could result from the formation of low soluble salts, i.e., the reaction product between aluminum and anions present in the test water. The test solution of each treatment was prepared at its respective nominal concentration: a certain amount of the test substance was added into the alga medium, and stirred (via magnetic stirrer) for 72h at 500 rpm in the dark followed by filtration using a 0.45μm nitrocellulose membrane.

Based on the results of the range-finding test, five nominal concentrations of 3.00 mg/L, 5.40 mg/L, 9.60 mg/L, 16.8 mg/L and 30.0mg/L (geometric mean measured concentrations of fluoride: 2.14 mg/L, 3.66 mg/L, 6.20 mg/L, 10.4 mg/L and 17.9 mg/L; those of aluminum: 0.0437 mg/L, 0.121 mg/L, 0.148 mg/L, 0.476 mg/L and 1.58 mg/L) were used in the definitive test. One control group of alga medium was run in addition to the treatment groups. Three replicates were included at each concentration as well as six replicates for the blank control .The test duration was 72h.

The mean biomass increase in the control cultures was 116, which is higher than the validity criterion of at least 16 within the 72-hour test period. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 24.9%, which was below 35%. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.43%, which was below 7%. Hence, the test was considered to be valid.

The measured concentrations of fluoride (F) analysed by ion chromatography (IC) for all treatment groups varied within ±20% from their initial concentrations (-7.00%-14.5%). The measured concentration of aluminum (Al) analysed by ICP-MS also varied within ±20% (-9.06%-15.1%) except the exposure concentration at 5.40 mg/L.

Since the test substance is a metal compound consisting of several elements (Li, Al and F), the results were calculated using the nominal concentrations in order to reflect the toxicity of the test substance. Based on average specific growth rate, the 72h-ErC10 value of the test substance to Pseudokirchneriella subcapitata was 7.57 mg/L (95% confidence limits: 1.46 mg/L–11.0 mg/L), and the 72h-ErC50 was 16.1 mg/L (11.3 mg/L–23.8 mg/L) . Based on yield, the 72h-EyC10 was 2.44 mg/L (0.177 mg/L– 4.17 mg/L), and the 72h-EyC50 was 6.96 mg/L (3.95 mg/L–11.4 mg/L).