Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-02-09 to 2011-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Nominal concentrations (main study): 0.5; 1.5; 4.9; 15.6 and 50 % v/v saturated solution, corresponding calculated test item concentrations (based on the analytical measurements): 0.2; 0.6; 1.8; 5.9 and 20.1 mg/L

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
Preparation of stock solution (0.5 mg/mL): -> Stock solution (0.1 mg/mL): 10 mg of the test item was weighed into a 100 mL volumetric flask, filled up to the mark with acetonitrile and thoroughly mixed.
Eluent: -> Acetonitrile : water +0.1% Formic acid - gradient
Dilution mixture: -> Acetonitrile: water at a ratio of 1: 1 (v/v)

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) (Printz-Starr)
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany

-Breeding Conditions:
The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days (The pre-culture was incubated for three days at this test.)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

22.6 – 23.2 °C (test flask)
22.0 - 23.8 °C (climate chamber)

pH:

7.77 – 9.30 within the course of the test

Nominal and measured concentrations:

Test concentrations were prepared by appropriate diluting of a saturated stock solution.
The following nominal concentrations were used in the main study: 0.5; 1.5; 4.9; 15.6 and 50 % v/v saturated solution.
The corresponding calculated test item concentrations (based on the analytical measurements) were: 0.2; 0.6; 1.8; 5.9 and 20.1 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (250 mL)
- Static test system
- Initial cells density: approximately 10.000 algal cells per mL test medium
- Control end cells density: approx. N= 79.17 × 10.000 cells/mL (mean value) after 72 hours
- No. of organisms per vessel: ca. 10.000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes; OECD medium (according to guideline)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201, 2006)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: 109 µE/m2/s, ensured with fluorescent lamps (with a spectral range of 400-700 nm) and checked periodically.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination cell numbers at 24, 48 and 72 hours by manual cell counting using a microscope with counting chamber.
- Morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours using a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations (main test): 0.5; 1.5; 4.9; 15.6 and 50 % v/v saturated solution, corresponding to calculated test item concentrations (based on analytical measurements) of 0.2; 0.6; 1.8; 5.9 and 20.1 mg/L, respectively.

Reference substance (positive control):

yes

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.5 mg/L

95% CI:

1.3 - 1.9

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.7 mg/L

95% CI:

0.6 - 0.9

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.7 mg/L

95% CI:

0.6 - 0.9

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2 mg/L

95% CI:

1.8 - 2.3

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.2 mg/L

95% CI:

3.7 - 4.9

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

95% CI:

1.9 - 2.6

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: biomass , growth rate and yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: biomass , growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): examined at 24, 48 and 72 h
- Unusual cell shape: examined at 24, 48 and 72 h
- Shape of the Algal Cells: The shape of the algal cells growing was obviously not affected.
- Cell Density in Control: In the control the cell density has increased from nominal N= 1.0E04 cells/mL at the start of the test (0 hrs) to N=79.17E04 cells/mL (mean value) after 72 hrs. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.

Results with reference substance (positive control):

Name: Potassium dichromate (K2Cr2O7)
The date of the last study with reference item Potassium dichromate was 07 - 10 December 2010.
- 72h ErC 50 = 1.40 mg/L, (95 % confidence limits: 1.29 – 1.53 mg/L)
- 72h EbC 50 = 0.92 mg/L, (95 % confidence limits: 0.85 – 0.99 mg/L)
- 72h EyC 50 = 0.92 mg/L, (95 % confidence limits: 0.85 – 1.00 mg/L)

The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test. Biological results are related to the geometric mean of the measured test item concentrations during the test.

 

Parameter	Value	95% confidence interval
NOEC 72h	0.6 mg/L
LOEC 72 h	1.8 mg/L
72h ErC50	4.2 mg/L	3.7 - 4.9
72h EbC50	2.2 mg/L	1.9 - 2.6
72h EyC50	2.0 mg/L	1.8 - 2.3
72h ErC10	0.7 mg/L	0.6 - 0.9
72h EbC50	1.5 mg/L	1.3 - 1.9
72h EyC50	0.7 mg/L	0.6 - 0.9

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test with Pseudokirchneriella subcapitata, the 72-h EC50 value based on growth rate was determined as 4.2 mg/L, based on biomass as 2.2 mg/L and based on yield was 2.0 mg/L. The overall NOEC was determined as 0.6 mg/L. The results are based on the measured geometric mean concentrations.

Executive summary:

Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was assessed in an aquatic toxicity test to algae according to EU method C.3 and OECD guideline 201(2006) under GLP compliance. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. Test concentrations were prepared by appropriate diluting of a saturated stock solution. In a pre-test nominal concentrations up to 100, in the main study nominal concentrations of 0.5; 1.5; 4.9; 15.6 and 50 % v/v saturated solution, corresponding to calculated test item concentrations (based on the analytical measurements) of 0.2; 0.6; 1.8; 5.9 and 20.1 mg/L, respectively. The 72 h-EC50 based on growth rate was determined to be 4.2 mg/L. The 72 h-EC50 based on yield was 2.2 mg/L. The overall NOEC was determined as 0.6 mg/L. The results are based on the measured geometric mean concentrations.

Description of key information

For Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc the 72 h-EC50 based on growth rate was determined to be 4.2 mg/L, the 72 h-EC50 based on yield 2.2 mg/L and the overall NOEC was determined as 0.6 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.2 mg/L

EC10 or NOEC for freshwater algae:

0.6 mg/L

Additional information

Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was assessed in an aquatic toxicity test to algae according to EU method C.3 and OECD guideline 201 (2006). Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. Test concentrations were prepared by appropriate diluting of a saturated stock solution. In a pre-test nominal concentrations up to 100, in the main study nominal concentrations of 0.5; 1.5; 4.9; 15.6 and 50 % v/v saturated solution, corresponding to calculated test item concentrations (based on the analytical measurements) of 0.2; 0.6; 1.8; 5.9 and 20.1 mg/L, respectively. The 72 h-EC50 based on growth rate was determined to be 4.2 mg/L. The 72 h-EC50 based on yield was 2.2 mg/L. The overall NOEC was determined as 0.6 mg/L. The results are based on the measured geometric mean concentrations.