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Ecotoxicological information

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:
2012-10-22 - 2012-10-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sample storage conditions before analysis: Room temperature, protected from moisture and light
Vehicle:
no
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

-Pseudokirchneriella subcapitata HINDÁK, SAG 61.81, Chlorophyta, Chlorophyceae

- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen

- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE•m-2•s-1 for 24 h per day.
- Culturing media: Nutrient medium Z according to LÜTTGE et al. (1994) :
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol Ca+Mg/L (Nominal)
Test temperature:
21 - 22.5 °C
pH:
8.15 - 9.26
Nominal and measured concentrations:
nominal: 0.00500 - 0.0158 - 0.0500 - 0.158 - 0.500 - 1.58 - 5.00 mg/L

measured: 0.00467 - 0.0153 - 0.0445 - 0.153 - 0.405 - 1.36 - 4.39 mg/L.

The concentrations of the active ingredient Isobornyl acrylate were analysed at all concentration levels at test start and test end via LC-MS. The measured concentrations at test start were in the range of 97 – 121 % of the nominal values. At the end of the test the test item gave recoveries of 67 – 80 %. All effect values are given based on nominal and geometric mean measured test item concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile headspace flasks (volume 119 mL) with aluminium tops and PTFE seals.

- Initial cells density: A three day old preculture was used as inoculum.
Nominal: 5000 - 10000 cells/mL
Measured: 5089 cells/mL

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
see table

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: see table
- Photoperiod: 24 h/d light
- Light intensity and quality: 60 - 120 µE • m-2 • s-1



TYPE AND FREQUENCY OF MEASUREMENT: Cell density was measured via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. The cell density was measured at the beginning of the test (after application) and every 24 h. Dilution water was used as background signal. The pH-value at the beginning of the test was measured out of one additional replicate of each test concentration and control. At the end it was measured from one pool per concentration of all replicates. The room temperature was measured continuously by a hygrothermograph. Light intensity was measured prior to test start. Microscopic evaluation of the cells at the start and at the end of the incubation was determined. Cells were checked for any unusual cell shapes, colour differences, chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of alga cells.

TEST CONCENTRATIONS

- Range finding study: see table below

Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.405 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.98 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 1.83 - 2.15
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.153 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.596 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.517 - 0.701
Details on results:
see table below
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA, batch number 1450864V, purity 100.3 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2012-04-10 to 2012-04-13. The reference item toxicity is in the valid range following test facility SOPS. For results see table.

Reported statistics and error estimates:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values was carried from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.


Results of the Range Finding Test (0 - 72 hours)

Nominal
test item concentration
[mg/L]

Growth rate inhibition
[%]

Yield inhibition
[%]

 10.0

100

100

   1.00

30

70

   0.100

7

23

   0.0100

4

16

Negative inhibitions = increase of growth

Main Test: Evaluation after 72 hours

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

 

Nominal test item concen-tration

Geometric mean measured
test item concen-tration

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

5.00

4.39

1

 

0.161

88.5

 

3153

 99.1

2

 

0.245

82.5

 

5531

 98.4

3

 

0.279

80.1

 

6655

 98.0

Mean

(+)

0.228

83.7

*

5113

 98.5

1.58

1.36

1

 

0.920

34.3

 

75308

 77.7

2

 

0.884

36.8

 

67104

 80.1

3

 

0.879

37.2

 

66050

 80.4

Mean

(+)

0.894

36.1

*

69487

 79.4

0.500

0.405

1

 

1.27

 9.47

 

222720

 34.0

2

 

1.29

 8.08

 

236468

 29.9

3

 

1.25

11.0

 

208623

 38.2

Mean

(+)1

1.27

 9.51

(+)

222604

 34.0

0.158

0.153

1

 

1.41

 -0.94

 

347656

 -3.03

2

 

1.40

 -0.26

 

337750

 -0.09

3

 

1.41

 -0.60

 

342735

 -1.57

Mean

(-)

1.41

 -0.60

(-)

342714

 -1.56

0.0500

0.0445

1

 

1.44

 -2.54

 

372199

-10.3

2

 

1.48

 -5.83

 

428047

-26.9

3

 

1.42

 -1.45

 

355371

 -5.32

Mean

(-)

1.45

 -3.27

(-)

385206

-14.2

0.0158

0.0153

1

 

1.46

 -4.10

 

397694

-17.9

2

 

1.44

 -2.72

 

375025

-11.1

3

 

1.42

 -1.46

 

355467

 -5.34

Mean

(-)

1.44

 -2.76

(-)

376062

-11.5

0.00500

0.00467

1

 

1.48

 -5.53

 

422681

-25.3

2

 

1.47

 -4.65

 

407092

-20.6

3

 

1.44

 -2.50

 

371596

-10.1

Mean

(-)

1.46

 -4.23

(-)

400456

-18.7

Control

1

 

1.35

 

 

286029

 

2

 

1.46

 

 

401822

 

3

 

1.45

 

 

384804

 

4

 

1.38

 

 

312096

 

5

 

1.43

 

 

362642

 

6

 

1.34

 

 

277203

 

Mean

 

1.40

 

 

337433

 

negative inhibitions = increase of growth

* = excluded to achieve normally distributed data

1) biologically not significant (inhibition < 10 %)

Validity criteria fulfilled:
yes
Remarks:
The study meets the validity criteria of the guideline: cell growth, test temperature, difference temperature from mean, pH value in the control, mean coefficients of variation of average spec. growth rates were fulfilled.
Conclusions:
In this study, Isobornyl acrylate was found to inhibit the growth of the freshwater green alga Pseudokirchnerella subcapitata after 72 h with the following effect values (measured test item concentrations):
EC50 (Growth rate): 1.98 mg/l; NOEC (Growth rate): 0.405 mg/L.
EC50 (Yield inhibition): 0.596 mg/L; NOEC (Yield inhibition): 0.153 mg/L

Executive summary:
The toxicity of Isobornyl acrylate to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 with GLP. The study was conducted under static conditions with an initial cell density of 5089 cells/mL.With regard to the volatility of the test item glass flasks without headspace were used to reduce losses of test item. Seven concentrations were tested in a geometrical series with a dilution factor of (nominal):0.00500 - 0.0158 - 0.0500 - 0.158 - 0.500 - 1.58 - 5.00 mg/L, corresponding to geometric mean measured test item concentrations of 0.00467 - 0.0153 - 0.0445 - 0.153 - 0.405 - 1.36 - 4.39 mg/L.

The concentrations of Isobornyl acrylate was determined in the fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via GC-FID. The measured concentrations of Isobornyl acrylate in the fresh media (0 hours) were in the range of 97 – 121 % of the nominal concentrations. After 72 hours they were in the range of 67 – 80 % of the nominal values at all tested concentration levels. All effect values given are based on the nominal and geometric mean measured concentrations.

The nominal EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours were 2.29 (2.12 – 2.48) mg/L and 0.351 (0.294 – 0.411) mg/L, respectively.The nominal NOEC-values for inhibition of growth rate and yield after 72 hours were 0.500 and 0.158 mg/L, respectively. The geometric mean measured EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 1.98 (1.83 – 2.15) mg/L and 0.596 (0.517 – 0.701) mg/L, respectively. The geometric mean measured NOEC-values for inhibition of growth rate and yield after 72 hours were 0.405 and 0.153 mg/L, respectively

 

Results Synopsis

 

Test Organism: Pseudokirchneriella subcapitata

Test Type: Algae Growth Inhibition Test, OECD TG 201, GLP

NOEC(Growth rate): 0.405 mg/L

EC50(Growth rate): 1.98 mg/L

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Description of key information

One reliable study for Isobornyl acrylate on algae with Pseudokirchneriella subcapitata acc. OECD 201 is available.

LC50(72h): 1.98 mg/l, NOEC (72h): 0.405 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:
1.98 mg/L
EC10 or NOEC for freshwater algae:
0.405 mg/L

Additional information