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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jan - 1 Feb 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: L-7038 Lot 2
- Expiration date of the lot/batch: 17 Jan 2002
- Purity: 97.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient conditions
- Stability under test conditions: stable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations monitored
- Sampling method: Three replicate test chambers were maintained for each treatment and control group. One additional replicate was also maintained for analytical sampling at 72 hours. Test solutions from the same concentration were pooled before sampling at 96 hours. The bulk test solution stocks fwere sampled at the initial time point. A sample was taken from all test concentrations and the control at test initiation, at approximately 72 hours, and at test termination.
- Sample storage conditions before analysis: samples stored in plastic vials and analyzed as soon as possible without storage
- Abiotic samples: To verify maintenance of the test substance concentration, two replicates were set up at the highest substance concentration but without algae. Measured test concentrations were determined at test initiation, at approximately 72 hours, and at test termination.
To verify maintenance of test substance concentration, a test vessel was set up at the highest substance concentration but without algae. Samples for analysis were taken at times zero, 24h and 72h.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 2-L primary stock solution was prepared in algal medium at a concentration of 10000 mg a.i./L. The primary stock solution was inverted at least 20 times to aid in the solubilization of the test substance. After mixing, the primary stock solution was proportionally diluted with algal medium to prepare 500 mL of the five other test concentrations (313, 625, 1250, 2500, 5000 mg a.i./L). All dilutions were inverted to mix.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none, test solutions were clear and colorless at start of test

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum
- Source (laboratory, culture collection): Internal stock at Wildlife International, Ltd. Inoculum originally obtained from University of Toronto, Canada.
- Age of inoculum: not reported
- Method of cultivation: Algal cells used in this test were obtained from Wildlife International, LtdCultures that had been actively growing in culture medium for at least two weeks prior to test initiation.

ACCLIMATION
- Acclimation period: none
- Culturing media and conditions: The algal cells were cultured and tested in freshwater algal medium. The pH of the medium was adjusted to 7.5 ± 0.1 using 10% HCl; the medium was sterilized by filtration (0.22 µm) prior to use.

MgCl2·6H2O, 12.16 mg/L
CaCl2·2H2O, 4.41 mg/L
H3BO3, 0.1855 mg/L
MnCl2·4H2O, 0.4154 mg/L
ZnCl2, 3.27 mg/L
FeCl3·6H2O, 0.1598 mg/L
CoCl2·6H2O, 1.428 mg/L
Na2MoO4·2H2O, 7.26 mg/L
CuCl2·2H2O, 0.012 mg/L
Na2EDTA·2H2O, 0.300 mg/L
NaNO3, 25.50 mg/L
MgSO4·7H2O, 14.70 mg/L
K2HPO4, 1.044 mg/L
NaHCO3, 15.0 mg/L

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
The 9478 mg a.i./L treatment group was maximally inhibited at the end of the 96-hour exposure period. Aliquots of the test solution were diluted with algal medium and cultured for six days. Based on the growth observed in the recovery phase, the effect on algal growth was found to be algistatic.

Test conditions

Test temperature:
23.8 - 24.7 °C
pH:
0 hours: 6.9 - 7.1
96 hours: 7.5 - 8.7
Nominal and measured concentrations:
Nominal: 0 (negative control), 313, 625, 1250, 2500, 5000, and 10000 mg/L
Measured: < LOQ, 285, 563, 1077, 2216, 4561, and 9478 mg/L (see Table 1)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL polycarbonate Erlenmeyer flasks plugged with foam stoppers and 100 mL of test or control algal medium
- Type: closed
- Agitation: Yes, the test chambers were shaken continuously at 100 rpm
- Initial cells density: 1.0E+04 cells/mL
- Control end cells density: 3.56E+06 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (ASTM Standard Guide 1218-90E)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified well water (reverse osmosis)

OTHER TEST CONDITIONS
- Photoperiod: Continuous cool-white fluorescent lighting (4300 ± 430 lux) measured using SPER Scientific Model 840006 light meter

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cell counts were conducted using a hemacytometer and microscope at 24 hour intervals

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5 661 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 5230-6067 mg a.i./L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
734 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0-2207 mg a.i./L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
5 733 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 5659-5817 mg a.i./L
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
1 674 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 1482-1839 mg a.i./L
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: After 96 hours of exposure, there were no signs of aggregation, flocculation, or adherence of the algae to the test flasks in the control or treatment groups. However, algal cells in the 2216, 4561, and 6478 mg a.i./L treatment groups appeared enlarged when compared to the negative control.
- Other: The 9478 mg a.i./L treatment group was maximally inhibited at the end of the 96-hour exposure period. Aliquots of the test solution were diluted with algal medium and cultured for six days. Based on the growth observed in the recovery phase, the effect on algal growth was found to be algistatic.
Reported statistics and error estimates:
After 72 hours of exposure, Dunnett’s test showed that growth rate was significantly reduced in all treatment groups (p = 0.05). After 96 hours of exposure, Dunnett’s test showed that growth rate was significantly reduced in the 2216, 4561, and 9478 mg a.i./L treatment groups (p = 0.05).

Any other information on results incl. tables

Table 2. Cell Densities over 96-Hour Exposure Period

 

Cell Densities (cells/mL)

Mean Measured Concentration (mg a.i./L)

Replicate

24 hours

48 hours

72 hours

96 hours

Negative Control

A

21000

168000

1200000

3735000

 

B

25000

184000

1045000

3420000

 

C

47000

185000

800000

3525000

285

A

29000

157000

625000

4005000

 

B

26000

152000

730000

3930000

 

C

36000

178000

710000

3825000

563

A

25000

136000

645000

3120000

 

B

30000

126000

625000

3195000

 

C

26000

154000

750000

3480000

1077

A

24000

133000

720000

3750000

 

B

17000

106000

465000

3180000

 

C

21000

118000

530000

3150000

2216

A

19000

88000

430000

2025000

 

B

14000

97000

330000

1845000

 

C

19000

101000

395000

1920000

4561

A

12000

51000

171000

850000

 

B

17000

80000

188000

870000

 

C

17000

56000

167000

860000

9478

A

17000

13000

16000

19000

 

B

4000

15000

16000

15000

 

C

15000

20000

12000

12000

Table 3. Mean Growth Rates and Percent Inhibition at 24-hour Time Intervals

 

Growth Rate

Mean Measured Concentration (mg a.i./L)

Replicate

0-24 hours

24-48 hours

48-72 hours

72-96 hours

0-72 hours

0-96 hours

Negative Control

A

0.0309

0.0866

0.0819

0.0473

0.0665

0.0617

 

B

0.0382

0.0832

0.0724

0.0494

0.0646

0.0608

 

C

0.0645

0.0571

0.0610

0.0618

0.0609

0.0611

285

A

0.0444

0.0704

0.0576

0.0774

0.0574

0.0624

 

B

0.0398

0.0736

0.0654

0.0701

0.0596

0.0622

 

C

0.0534

0.0666

0.0576

0.0702

0.0592

0.0619

563

A

0.0382

0.0706

0.0649

0.0657

0.0579

0.0598

 

B

0.0458

0.0598

0.0667

0.0680

0.0574

0.0601

 

C

0.0398

0.0741

0.0660

0.0639

0.0600

0.0610

1077

A

0.0365

0.0713

0.0704

0.0688

0.0594

0.0617

 

B

0.0221

0.0763

0.0616

0.0801

0.0533

0.0600

 

C

0.0309

0.0719

0.0626

0.0743

0.0551

0.0599

2216

A

0.0267

0.0639

0.0661

0.0646

0.0522

0.0553

 

B

0.0140

0.0807

0.0510

0.0717

0.0486

0.0544

 

C

0.0267

0.0696

0.0568

0.0659

0.0511

0.0548

4561

A

0.0076

0.0603

0.0504

0.0668

0.0394

0.0463

 

B

0.0221

0.0645

0.0356

0.0638

0.0407

0.0465

 

C

0.0221

0.0497

0.0455

0.0683

0.0391

0.0464

9478

A

0.0221

-0.0112

0.0087

0.0072

0.0065

0.0067

 

B

0.000

0.0551

0.0027

-0.0027

0.0065

0.0042

 

C

0.0169

0.0120

-0.0213

0.0000

0.0025

0.0019

The mean CV for section specific growth rates in the control was 29%, and the CV of average specific growth rates during the whole test period in control was 4.4%

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control cell density increased by an average factor of >16 within 2 days, the mean CV for section specific growth rates in the control was < 35% (29%), and the CV of average specific growth rates during the whole test period in control was < 7% (4.4%).
Conclusions:
The 72-hour EC50 (growth rate) of PFBSK+ to Pseudokirchneriella subcapitata was 5661 mg a.i./L. The corresponding 72-hour EC10 (growth rate) was 734 mg a.i./L. Test conducted per OECD 201.
Executive summary:

The toxicity of PFBSK+ to the green algae, Pseudokirchneriella subcapitata, was assessed according to the OECD 201 method. An exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met.

The 72-hour ErC50 of PFBSK+ to Pseudokirchneriella subcapitata for growth rate was 5661 mg a.i./L (5230-6067 mg a.i./L). The corresponding 72-hour ErC10 was 734 mg/L (95% CI: 0-2207 mg/L). Based on the growth observed in the recovery phase, the effect on algal growth was found to be algistatic.

The study was performed in accordance with internationally-accepted test guidelines under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis.