Reaction products of 1,4-Benzenedimethanol and 1-naphthol

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:

06 May 2013 to 09 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE).

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-7 The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000 & Ref. No.13-Seisan-3986 on 10 October 2001).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report

Analytical monitoring:

yes

Remarks:

HPLC-UV analysis

Details on sampling:

The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Vehicle:

no

Details on test solutions:

Because the test item is very poorly soluble in water, a test solution was prepared using a saturated solution method. A supersaturated test item stock solution (nominally 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30°C and then was equilibrated for about 24 hours at the test temperature. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100 % v/v saturated solution.
As limit test was carried out, further dilution of stock solution was not performed.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Initial cell number: The initial cell number in the test cultures was 104 cells/mL.
Pre-culturing: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 107 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

Carried out in accordance to stud guidelines

Post exposure observation period:

Not specified

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.3 – 22.6 °C measured in the flask and between 22.1 and 23.1 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the experiment, in the control and in the used test concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.76 – 9.38 during the experiment.

Nominal and measured concentrations:

Because toxic effect was not observed in the preliminary range-finding test, only one test concentration at the solubility level of the test item in the test medium (100 % v/v saturated solution) and one control group was tested in a limit test.

Details on test conditions:

Light Intensity
The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 8116 lux (equivalent to 110 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel

DESCRIPTION OF THE TEST PROCEDURE
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.
The test was performed with six replicates per test concentration and six replicates in the control group. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Preliminary Range Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
During the formulation procedure the test solutions were prepared by similar method described above, except for test solution of 1 % v/v saturated solution and 0.1% v/v saturated solution. These solutions were prepared by appropriate dilution of test solutions of 10 % v/v saturated solution and 1 % v/v saturated solution respectively.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

not specified

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

not specified

Details on results:

The cell density in the control cultures increased by a factor of 67.17 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 7.86 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.52 %.
All validity criteria were met, therefore the study can be considered as valid.

AVERAGE SPECIFIC GROWTH RATES
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the average specific growth rates were not statistically significantly different from the untreated control value. The No Observed Effect Concentration (NOEC) was determined as the 100 % v/v saturated solution.

AREAS UNDER THE GROWTH CURVES
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the areas were not statistically significantly different from the untreated control value. The No Observed Effect Concentration (NOEC) was determined as the 100 % v/v saturated solution.

YIELD
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value. The No Observed Effect Concentration (NOEC) was determined as the 100 % v/v saturated solution.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 13/019-022AL) with the reference item Potassium dichromate is (Batch Number: 0769128): 05 - 08 February 2013.
The 72h ErC 50 : 0.87 mg/L, (95 % confidence limits: 0.79 – 0.96 mg/L)
The 72h EbC 50 : 0.48 mg/L, (95 % confidence limits: 0.44 – 0.53 mg/L)
The 72h EyC 50 : 0.45 mg/L, (95 % confidence limits: 0.41 – 0.50 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test item were determined from the raw data.
Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) 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 2 Sample t-Test (α = 0.05) by TOXSTAT software.

Results of the Preliminary Range-Finding Test

 

% v/v saturated solution	Untreated control	0.1	1.0	10.0	100.0
Average of cell number at 72 hours (x 10^4cells/mL)	73.33	73.00	71.00	68.00	67.50

Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

 

Test group	Areas under the Growth Curves (A) and % inhibition of A
	0 – 24 h		0 – 48 h		0 – 72 h
	μ	%	μ	%	μ	%
Control	42.0	0.0	288.0	0.0	1286.0	0.0
100 % v/v saturated solution	42.0	0.0	290.0	-0.7	1266.0	1.6

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

 

Test group	Areas under the Growth Curves (A) and % inhibition of A
	0 – 24 h		0 – 48 h		0 – 72 h
	μ	%	μ	%	μ	%
Control	42.0	0.0	288.0	0.0	1286.0	0.0
100 % v/v saturated solution	42.0	0.0	290.0	-0.7	1266.0	1.6

Yield (Y) and Percentage Inhibition of Y during the Test Period

 

Test group	Yield (Y) and % inhibition of Y (0 – 72 h)
	Y	%
Control	66.2	0.0
100 % v/v saturated solution	64.2	3.0

pH-Values in the Test Media at the Start and End of the Test

 

Test Group	pH-values
	Start	End
Control	7.76	9.38
		9.37
		9.32
		9.35
		9.27
		9.37
100 % v/v saturated solution	7.84	9.19
		9.27
		9.22
		9.21
		9.23
		9.14

Temperature in the Climate Chamber and in the Test Media during the Test

 

Parameter		Exposure Time
		Day 0	Day 1	Day 2	Day 3
Temperature (ºC) measured in flask		22.3	22.6	22.5	22.6
Min/max temperature (ºC) of the climate chamber	Max	22.8	23.1	22.9	22.9
	Min	22.1	22.3	22.3	22.4

Cell Number (x 10⁴cell/mL) determined in the Main Experiment

 

Test group	Number of cells
	0 h	24 h	48 h	72 h
Control	1	4	17	68
	1	4	16	66
	1	5	20	69
	1	5	18	65
	1	4	17	67
	1	5	20	68
Mean	1.00	4.50	18.00	67.17
SD	0.0	0.5	1.7	1.5
100 % v/v saturated solution	1	5	17	59
	1	4	17	66
	1	5	20	67
	1	5	18	66
	1	4	19	65
	1	4	18	68
Mean	1.00	4.50	18.17	65.17
SD	0.0	0.5	1.2	3.2

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, the test item SN-475N had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.

Executive summary:

The effect of SN-475N test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

Because no significant inhibition of algal growth was observed during the preliminary range-finding test, a limit test was carried out using only one concentration at the solubility level of the test substance in the test medium (100 % v/v saturated solution) and one control group.

Test concentration was analytically determined at the start and at the end of the experiment, however the measured concentration was below the Limit of Quantification (LOQ = 2.0 mg/L) also at the start and at the end of the test, after increasing the concentration by a factor of 25 by freeze-drying method. Therefore it can be stated that the solubility level of the test item is lower than 2.0/25=0.08 mg/L.

The test design included six replicates at test concentration and six replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item were determined directly from the raw data.

Under the conditions of this algal growth inhibition test the observed endpoints for the effect of SN-475N were the following:

The 72h EbC50 value (biomass): > 100 % v/v saturated solution

The 72h ErC50 value (growth rate): > 100 % v/v saturated solution

The 72h EyC50 value (yield): > 100 % v/v saturated solution

The No-Observed Effect Concentration (NOEC): 100 % v/v saturated solution

The Lowest Observed Effect Concentration (LOEC): > 100 % v/v saturated solution

Based on the results of this study, the test item SN-475N had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.

Description of key information

The effect of SN-475N test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

The ErC50, EbC50 and EyC50 values of the test item were determined directly from the raw data.

Based on the results of this study, the test item SN-475N had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Under the conditions of this algal growth inhibition test the observed endpoints for the effect of SN-475N were the following:

The 72h EbC50 value (biomass): > 100 % v/v saturated solution

The 72h ErC50 value (growth rate): > 100 % v/v saturated solution

The 72h EyC50 value (yield): > 100 % v/v saturated solution

The No-Observed Effect Concentration (NOEC): 100 % v/v saturated solution

The Lowest Observed Effect Concentration (LOEC): > 100 % v/v saturated solution