2,2'-[6-(4-methoxyphenyl)-1,3,5-triazine-2,4-diyl]bis{5-[(2-ethylhexyl)oxy]phenol}

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start 08 January, 2014; Completion 17 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Molecular formula: C38H49N3O5Molecular weight: 627.8CAS Number: 187393-00-6Description: Pale yellow powderBatch: 0M000254477Purity/Composition: 98%Test substance storage: At room temperature in the darkStable under storage conditions until 28 October 2015 (retest date)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling: Frequency At the beginning and at the end of three intervals of 48 hours (nominal days 0 and 2, 8 and 10, 14 and 16)Volume: 2.0 mlStorage: Samples not analysed on the day of sampling were stored in a freezer until analysis.At the end of the refreshment period, the replicates were pooled at each concentration before sampling.Additionally, reserve samples of 2.0 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months.

Test solutions

Vehicle:

yes

Details on test solutions:

An acetone1 stock was prepared at a loading rate of 10 g/l applying a short period (approximately 15 minutes) of magnetic stirring to accelerate dissolution of the test substance. The obtained stock was clear and slightly yellow. A stock containing 1.0 g Escalol S per litre acetone was prepared by diluting the 10 g/l stock in acetone. The obtained stock was clear and colourless. Stocks were prepared weekly and stored in the freezer until use.Test solutions were prepared freshly before each renewal. 100 μl of the respective stocks were spiked per 1 litre of test medium and magnetically stirred for a period of 15 minutes to ensure homogenous distribution. The obtained test solutions were clear and colourless (i.e. 0.1 mg/l) and hazy (i.e.1.0 mg/l). A solvent control was prepared in a similar way, i.e. by spiking 100 μl acetone to one litre test medium and stirring for approximately 15 minutes.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISMSpecies: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), atleast third generation, obtained by acyclical parthenogenesis under specified breeding conditions.Source: In-house laboratory culture with a known history.Reason for selection: This system has been selected as an internationally accepted invertebrate species.Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.CHARACTERISTICS: To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.BREEDING CONDITIONS:Start of each batch with newborn daphnids, i.e. less than 3 days old, by placing them individually in 50 ml M7-medium. Maximum age of the cultures 4 weeks. Monitoring of the individual cultures Three times a week the young are counted and the parental daphnids are transferred to new media. Renewal of the cultures after 7 days of cultivation half of the medium twice a week. Temperature of medium 18-22°C. Feeding Daily, a suspension of fresh water algae. Validity of the cultures Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.Medium: Medium M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

179 and 214 mg CaCO3 per liter

Test temperature:

18 and 20°C

pH:

7.6 to 8.8

Dissolved oxygen:

7.4 to 11 mg/l

Nominal and measured concentrations:

Nominal concentrations: 0.1 and 1.0 mg/LMean measured concentrations: 0.060, 0.70 mg/L

Reference substance (positive control):

not required

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 4

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 5

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Any other information on results incl. tables

Table 1: Survival of parental daphnids during the 21-day exposure period in the second reproduction test

Nominal day	Concentration^1Escalol S (mg/l)
	Blank control	Solvent control	0.060	0.70
0	10	10	10	10
8	10	10	10	10
9	10	10	10	10
10	10	10	10	10
11	9	10	10	10
12	9	10	10	10
14	9	10	9	10
15	9	10	9	10
16	9	10	7	10
17	9	10	7	9
18	9	10	7	9
20	9	10	7	9
21	8	10	7	9

1. Average exposure concentration

Table 2: Group mean cumulative number of juveniles per surviving parent and reduction of reproduction at the end of the second reproduction test

	Concentration^1Escalol S (mg/l)
	Blank control	Solvent control	0.060	0.70
Mean:	88	99	110	109
Std.Dev.:	18	38	12	15
n:	8	10	7	9
CV:	20	39	11	13
% Reduction^2:	-	-	-11	-10

1. Average exposure concentration

2. Calculated relative to the solvent control. Negative numbers indicate an increase of reproduction.

Table 3: Group mean body lengths and reduction of growth of parental daphnids at the end of the second reproduction test

Concentration^1Escalol S (mg/l)	Mean	Std. Dev.	n	% Reduction^2
Blank control	3.93	0.27	8	0.0
Solvent control	3.96	0.49	10	0.0
0.06	4.13	0.13	7	-4.4
0.70	4.12	0.21	9	-4.1

1. Average exposure concentration

2. Calculated relative to the solvent control. Negative numbers indicate an increase of body length.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study Escalol S did not affect reproduction of Daphnia magna at an average exposure concentration of 0.70 mg/l after 21 days of exposure (NOEC). This concentration is considered to be above solubility of Escalol S in test medium.

Executive summary:

The study procedures described in this report were based on theOECD guidelines for Testing of Chemicals: Guideline No. 211, 2012. In addition,the procedures were designed to meet the test methods and validity criteria of theISO International Standard 10706, 2000, the Commission Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

The batch of Escalol S tested was a pale yellow powder with a purity of 98%.

Test solutions were prepared freshly before each renewal by spiking test medium with pre-dissolved acetone stocks. The obtained test solutions were clear and colourless (i.e. 0.1 mg/l) and hazy (i.e. 1.0 mg/l) indicating that, at least, the highest concentration was above solubility in test medium.

The reproduction test was performed in a semi-static system, included 10 vessels per test group (two concentrations, blank and solvent control). Each of the vessels contained one neonate (<24h old)Daphnia magnain 50 ml test medium. The nominal concentrations of Escalol S tested were 0.1 and 1.0 mg/l.

The study duration was 21 days and the test solutions were renewed every 48 hours. The daphnids were fed on a daily basis with aChlorella pyrenoidosasuspension. Every workday the condition of the parental daphnids was recorded, during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test the lengths of the surviving parental daphnids were measured.

During the study samples for analyses were taken at the beginning and the end of three intervals of 48 hours.

The actual concentrations measured in nominally 0.1 and 1.0 mg/l were at the level of 53 to 88% of nominal. The measured concentrations were between 65 and 123% of initial at the end of each interval. The average exposure concentrations were calculated to correspond to 0.060 and 0.70 mg/l, respectively.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

No treatment-related mortality of parental daphnids was observed during the test.

The average cumulative number of young per female after 21 days was 88 and 99 in the blank and solvent control, respectively. 

No reduction or delay in reproduction was observed at any of the concentrations tested.

The growth of daphnids was not affected by the test substance.

Escalol Sdid not affect reproduction ofDaphnia magnaat an average exposure concentration of 0.70 mg/l after 21 days of exposure (NOEC). This concentration is considered to be above solubility of Escalol S in test medium.