Reaction mass of 1-Hydroxyoctane, 2-methanoate and 1,2-Dihydroxyoctane, dimethanoate and 2-Hydroxyoctane 1-methanoate and 1,2-Dihydroxyoctane

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:

31 Mar - 30 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office for the Environment (FOEN)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: duplicate samples were taken at the start and at the end of each test medium renewal period from all tested concentrations and from the control
- Sampling method: the contents of the respective replicates were combined prior to sampling at the aged test media
- Sample storage conditions before analysis: samples were stored deep-frozen (at about -20 °C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAFs were prepared by adding of individual amount of test item for each loading rate, solutions were stirred slowly 24 h to dissolve a maximum amount of the different compounds of the test item. Then the lower equilibrated water phases were carefully removed from each stirring vessel through the tap at the bottom of the vessels while the non-dissolved test item floating on the water surface remained in the stirring vessels.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): laboratory culture
- Age of inoculum (at test initiation): 4 d, exponential growth phase (determined by an electronic particle counter)
- Method of cultivation: algae were cultivated under the test conditions for 4 d

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

-

Test temperature:

23 °C

pH:

7.3 - 9

Dissolved oxygen:

-

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal: 1, 3.2, 10, 32 and 100 mg/L (loading rates, WAF)
Measured at test initiation: < LOQ = 0.7 mg/L, 1.0, 3.7, 19.8 and 91.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 75 mL Erlenmeyer flasks (covered with a glass dish)
- Fill volume: 30 mL
- Initial cells density: 5000 cells/mL (0.74 x 10^4 relative fluorescence units)
- Control end cells density: 158.6 x 10^4 (SD: 8.6) relative fluorescence
units
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: pH was measured in each treatment at the start and end of the test. Temperature was monitored continuously and the appearance of the test media was controlled daily.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 76 μE s^-1 m^-2, LED light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: 200 μL of the algal suspension was withdrawn and duplicates were measured daily by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd) at an excitation of 440 nm and emission of 680 nm
- Other: shape and size of the algal cells were visually inspected at the end of the test in the control and 32 mg test item/L This loading rate was chosen since the algal cell density was too low for a reliable examination at the loading rate 100 mg/L.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 100 mg/L, 1:10 dilution and 1:100 dilution
- Results used to determine the conditions for the definitive study: % inhibition of average growth after 72 h was 16% at the loading rate of 100 mg/L, 3.2% at the 1:10 dilution and 0.6% at the 1:100 dilution

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 45 – 47

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 22 – 24

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: 13 - 15

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

2.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: 2.2 - 3.2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes (biomass increased by a factor of 215 over 72 h)
- Observation of abnormalities (for algal test): microscopic examination of the shape and size of the algal cells at the end of the test showed no difference between the algae growing at the loading rate of 32 mg/L and those of the control.
- Unusual cell shape: no
- Flocculation: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 (72 h): 0.74 mg/L

Reported statistics and error estimates:

The 72-hour EL10 and EL50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression.
For the determination of the LOEL and NOEL, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t-test.
Statistical analysis was performed using ToxRat Professional®.
The values for the validity criteria of the test were also calculated by the statistic program ToxRat Professional®.

Since water accommodated fractions of the test item were tested, all reported biological results were based on the loading rates of the test item.

The measured concentrations at the end of the test were in the range of 0 and 40% of the initially measured values.

Table 1: Analytical results.

Sampling Day / Sample Age [d/h]	Loading Rate of Test Item [mg/L]	Determined Concentration of Test Item [mg/L]
0/0 (fresh)	Control	< 0.7
	1	< 0.7
	3.2	1.03
	10	3.73
	32	19.8
	100	91.6
3/72 (aged)	Control	< 0.7
	1	< 0.7
	3.2	< 0.7
	10	< 0.7
	32	< 0.7
	100	36.3

The pH increased in the control from 7.3 at test start to 9.0 at test end; according to the OECD guideline, the pH of the control medium should not increase by more than 1.5 units during the test. The increase in pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being shaken continuously during the test.

However, since all validity criteria concerning the growth are fulfilled the test is considered to be valid.

Table 2: pH values.

	pH
Loading rate [mg/L]	Start	End
Control	7.3	9
1	7.4	9
3.2	7.4	9
10	7.4	8.9
32	7.4	8.3
100	7.3	7.4

Table 3: % Inhibition of growth rate and yield during the test (average).

Loading rate [mg/L]	24 h	48 h	72 h
	% inhibition of growth rate
Control	0	0	0
1	4.5	-2.7	1
3.2	7.6	2.8	3.8
10	5.8	7.4	7.7
32	40.9	27.2	24.9
100	101.6	94.9	92.1
	% inhibition of yield
Control	0	0	0
1	9.5	-10.5	5.1
3.2	15.1	10	18.4
10	11.7	24.3	34.3
32	60.7	64.7	74
100	100.3	99.4	99.8

Validity criteria fulfilled:

yes

Remarks:

except of a high increase of pH in the control after 72 h due to rapid growth of algae

Conclusions:

The test item showed toxic effects on on the growth of the freshwater green algal species Pseudokirchneriella subcapitata resulting in an ErL10 (72 h) = 23 mg/L and ErL50 (72 h) = 46 mg/L based on loading rates.

Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to OECD 201 (2011) and GLP.

In order to assess the toxicity of the substance as a mixture of four constituents to algae, water accommodated fractions (WAFs) with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were tested.

Following the slow stirring method, five individual solutions with the corresponding test item loading rate were slowly stirred for 24 hours to dissolve a maximum amount of the different compounds of the test item in the solutions. The stirring period was based on the pre-experiment. The analytical measurements of the soluble components of the test item were based on two main components of the test item, which represent approximately 60% of the test item.

During the test a decrease of the analyzed components in the test media occurred. The measured concentrations at the end of the test were in the range of 0 and 40% of the initially measured values.

Since WAFs of the test item were tested, all reported biological results were based on the loading rates of the test item.

Description of key information

EL50 (72 h) = 46 mg/L and EL10 (72 h) = 23 mg/L (WAF), based on growth rate of Pseudokirchneriella subcapitata (OECD 201, GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:

46 mg/L

EC10 or NOEC for freshwater algae:

23 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to freshwater algae. The study was conducted according to OECD 201 and GLP using Pseudokirchneriella subcapitata as the test species. The test organism was exposed to the loading rates of 1, 3.2, 10, 32 and 100 mg/L (WAF) for 72 h. Based on growth rate inhibition the test resulted in an ErL50 (72 h) of 46 mg/L and an ErL10 (72 h) of 23 mg/L (loading rate, WAF). Since water accommodated fractions of the test item were tested, the reported biological results were based on the loading rates of the test item.