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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:
2019-05-13 to 2019-05-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Sampling and analysis

Analytical monitoring:
no
Remarks:
No analytical monitoring was carried out due to the low solubility of the test item.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble and multi-component test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents.
Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture).
Exposure are expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fraction
One water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L. The loading levels are based on the results of a preliminary range finding test. An appropriate amount of the test item was weighed out and transferred into a measuring flask with an appropriate amount of the test media (see Table 1 and Table 2). This dispersion was shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour at room temperature, the aqueous phase or WAF was removed by siphoning (from the approximate centre of the glass flask). The resulting water accommodated fraction (WAF) was used in a limit test.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4 (axenic)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Age of inoculum (at test initiation): a four days old preculture
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567–5130 lux for 24 h per day
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
river water total hardness [mg CO3/l]: 184
Test temperature:
Nominal range: 21 - 24°C, controlled at ± 2°C
pH:
start: 8.23 (test 100 mg/l) 8.24 (control); end: 8.33 (test 100 mg/l) 8.34 (control)
Dissolved oxygen:
14.86 mg O2/L (river water)
Conductivity:
987 µS/m (river water)
Nominal and measured concentrations:
100 mg/L loading rate water accomodated fraction
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, provided with cotton wool plugs. The test container were pretreated with the appropriate test solutions for 72 hours under test conditions. Before the start of the exposure phase, the test container were emptied and refilled with freshly prepared test solutions.
- Fill volume: 100 mL
- Initial cells density: nominal: approximately 5 x 10 E03 E04 cells/mL; actual: 5416 cells/mL
- Control end cells density: 1256062 (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no, natural river water, for details see section "any other information on materials and methods"

TEST MEDIUM / WATER PARAMETERS
river water as specified in section "any other information on materials and methods"

OTHER TEST CONDITIONS
- Sterile test conditions: The natural river water was stored at 15 ± 2 °C and at - 18± 2 °C for at least 24 hours. Freezing was found to be suitable to minimize the content of vital natural algae cells of the water as well as to reduce microbial (bacterial) activity
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
Range finding study
- Test concentrations: 1.00 - 10.0 - 100 mg/L nominal
- Results used to determine the conditions for the definitive study:
Growth Rate Inhibition: 100: 1%, 10.0: -1%, 1.00: 0% each concentration mg/l nominal
Yield Inhibition: 100: 4%, 10.0: -3%, 1.00: -2% each concentration mg/l nominal
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal test item loadings
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal test item leadings
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.
Results with reference substance (positive control):
- Results with reference substance valid?
- ErC50: 0.701 (95 % c.l. 0.660 - 0.750 mg/L); valid range: 0.752 ± 0.534
- EyC50: 0.353 (95 % c.l. 0.312 - 0.394), valid range: 0.405 ± 0.308
- Other: tested from 2019-03-05 to 2019-03-08
Reported statistics and error estimates:
Statistical Significance (NOEL/LOEL) – Growth Rate

t-test

Normality Test (Shapiro-Wilk): Passed (P = 0.222)
Equal Variance Test (Brown-Forsythe): Passed (P = 0.860)

The result of the equal variance test indicates the likelihood that the two groups are sampled from populations with equal variances. but does not guarantee the equality or inequality of the two variances.

Group Name N Missing Mean Std Dev SEM
Control 6 0 1.815 0.0237 0.00969
100 mg/L 6 0 1.816 0.0188 0.00767

Difference of means -0.001000
Use the results of Welch's test. where equal variances are not assumed. if the equality of the population variances of the two groups is in doubt.

Equal Variances Assumed (Student's t-test):

t = -0.0809 with 10 degrees of freedom.

95 percent two-tailed confidence interval for difference of means: -0.0285 to 0.0265
Two-tailed P-value = 0.937

The difference in the mean values of the two groups is not great enough to reject the possibility that the difference is due to random sampling variability. There is not a statistically significant difference between the input groups (P = 0.937).

Equal Variances Not Assumed (Welch's t-test):

t = -0.0809 with 9.501 degrees of freedom.

95 percent two-tailed confidence interval for difference of means: -0.0287 to 0.0267
Two-tailed P-value = 0.937

The difference in the mean values of the two groups is not great enough to reject the possibility that the difference is due to random sampling variability. There is not a statistically significant difference between the input groups (P = 0.937).

The program states no significant difference for the limit loading rate compared to the control.  

Any other information on results incl. tables

Cell Densities

Nominal test item loading

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

100

1

5416

33137

418524

1261075

2

5416

40989

411057

1245011

3

5416

39689

443906

1266479

4

5416

38993

452649

1395043

5

5416

40619

416304

1186595

6

5416

38472

453330

1207771

Mean

5416

38650

432628

1260329

Control

1

5416

33709

328758

1256638

2

5416

32258

289512

1247340

3

5416

36320

269651

1271534

4

5416

34130

296961

1267591

5

5416

28529

215889

1110063

6

5416

37800

326480

1383206

Mean

5416

33791

287875

1256062

Evaluation after 72 hours

           Statistically significant differences of growth rates and yield compared to
           control values are marked (+), not significant differences are marke

Nominal test item loading

Replicate

Growth rate

Growth rate inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.82

0

 

1255659

0

2

         

1.81

0

 

1239595

1

3

1.82

0

 

1261063

-1

4

 

1.85

-2

 

1389627

-11

5

 

1.80

1

 

1181179

6

6

 

1.80

1

 

1202355

4

Mean

(-)

1.82

0

(-)

1254913

0

Control

1

 

1.82

 

 

1251222

 

2

 

1.81

 

 

1241924

 

3

 

1.82

 

 

1266118

 

4

 

1.82

 

 

1262175

 

5

 

1.77

 

 

1104647

 

6

 

1.85

 

 

1377790

 

Mean

 

1.82

 

 

1250646

 

Negative values = growth stimulation

Section-by-Section and Average Specific Growth Rates of the Control Group

           (0 – 72 hours)

 

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.83

2.28

1.34

1.82

0.469

25.8

18.5

2

1.78

2.19

1.46

1.81

0.368

20.3

3

1.90

2.01

1.55

1.82

0.238

13.1

4

1.84

2.16

1.45

1.82

0.357

19.6

5

1.66

2.02

1.64

1.77

0.217

12.2

6

1.94

2.16

1.44

1.85

0.366

19.8

 

 

 

Mean

1.82

 

 

 

 

 

SD ±

0.02

 

 

 

 

CV [%]

1.29

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
72 h NOEL of Ceramide VI to the green alga Pseudokirchneriella subcapitata based on growth rate and yield inhibition are 100 mg/l water accomodated fraction (nominal).
72h LOEL, ErL10, ErL50 based on growth rate and yield inhibition are > 100 mg/l WAF (nominal).
Executive summary:

In a 72 hour toxicity study according to OECD Guideline 201 (2011), the cultures of Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4 (axenic) were exposed to Ceramide VI at a nominal limit concentrations of 100 mg/L water accommodated fraction in natural river water under static conditions. The concentration was not measured due to low water solubility.

There was no statistically significant growth inhibition in the treated algal culture as compared to the control.

NOEL (based on growth rate) and NOEL based on yield inhibition were 100 mg/L WAF (nominal).

LOEL, EL10, EL50 based on growth rate or yield inhibition are > 100mg/L WAF (nominal).

Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for the algal growth inhibition test in Pseudokirchneriella subcapitata.

 

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4 (axenic) 

Test Type: Static

Exposition: water accommodated fraction

 

72h NOEL (based on growth rate and yield inhibition): 100 mg/L WAF (nominal).

72h LOEL, EL10, EL50 (based on growth rate or yield inhibition): > 100mg/L WAF (nominal).