reaction mass of 2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane and cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane and trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 - 09 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane

Molecular formula:

C17H29ClO5

IUPAC Name:

2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane

Constituent 2

Reference substance name:

cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Cas Number:

1197197-64-0

Molecular formula:

C14H26O4

IUPAC Name:

cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Constituent 3

Reference substance name:

trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Cas Number:

158307-92-7

Molecular formula:

C14H26O4

IUPAC Name:

trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

impurity 1

Reference substance name:

[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methanol

Molecular formula:

C11H20O3

IUPAC Name:

[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methanol

impurity 2

Reference substance name:

[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methanol

Molecular formula:

C14H25ClO4

IUPAC Name:

[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methanol

impurity 3

Reference substance name:

2-[[3-chloro-2-[[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methoxy]propoxy]methyl]oxirane

Molecular formula:

C20H34Cl2O6

IUPAC Name:

2-[[3-chloro-2-[[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methoxy]propoxy]methyl]oxirane

impurity 4

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No.of test material: TX7E5501
- Physical state: pale yellow liquid
- Purity: 87.6%
- Expiration date of the lot/batch: 26 May 2022
- Storage condition of test material: ambient

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All; sampled at test initiation (0 hours) and at test termination (72 hours).
- Sampling method: Samples were processed immediately for analysis. Samples collected at test initiation (0 hour) were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution into the test chambers. At exposure termination (72 hours), samples were collected from the pooled replicates from each treatment and control group and the single abiotic replicate in the highest treatment group. At each sampling interval, 5.0 mL of test solution was added to 20 mL glass scintillation vials containing 5.0 mL of 0.2% formic acid in methanol.
- Sample storage conditions before analysis: not stored

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The 2.0, 6.3, 20, and 64 mg/L test solutions were prepared by weighing the required mass of Heloxy 107 on a Teflon disc and quantitatively adding the disc to 4L aspirator bottles containing 4L of freshwater AAP medium. The 2.0, 6.3, 20, and 64 mg/L test solutions were stirred with Teflon-coated stir bars overnight. On the following day, the aliquots of the 2.0 mg/L test solution were diluted with freshwater AAP medium to prepare test solutions at nominal concentrations of 0.19 and 0.61 mg/L. All test solutions appeared clear and colorless and otherwise unremarkable at the time of preparation.
- Controls: freshwater AAP medium without test substance
- Evidence of undissolved material: No

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: not reported
- Source (laboratory, culture collection): Original algal cultures were obtained from the University of Texas at Austin, and have been maintained in culture medium at EAG Laboratories, Easton, Maryland since June 2017.
- Age of inoculum (at test initiation): Algal cells were inoculated four days before the test from an actively growing culture medium which had been maintained under similar environmental conditions as used in this test for at least two weeks prior to test initiation
- Method of cultivation: Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified EAG Laboratories-Easton well water (NANOpure® water). The test medium then was prepared by adding appropriate volumes of the stock nutrient solutions to NANOpure® water. The pH of the medium was adjusted to 7.5 ± 0.1 with 10% hydrochloric acid and/or 0.1N sodium hydroxide, as needed, at the time of preparation. The medium was sterilized by filtration (0.22 μm) and stored refrigerated prior to use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.54 - 24.72 °C

pH:

7.3 - 9.8

Nominal and measured concentrations:

Nominal: 0.19, 0.61, 2.0, 6.3, 20 and 63 mg formulation/L
Measured: see Table 1. Nominal concentrations were used to determine effect concentrations. Test substance was a mixture, and the two predominant components showed reasonable stability over the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile 250-mL glass Erlenmeyer flasks plugged with sterile foam stoppers
- Type: closed
- Fill volume: 100 mL
- Aeration: continuous shaking at 100 RPM
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 302E+04 cells/mL
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes, AAP medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified well water (NANOpure®)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5,610 to 6,540 lux (measured at test solution level at five locations surrounding the test flasks at test initiation using a SPER Scientific 840006C light meter)

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: electronic particle counter (Z2; Coulter Electronics, Inc.), appearance of cells. Cell standards were quantified by hemacytometer count before being used in the electronic particle counter.
- Recovery phase: At the end of the 72-hour exposure period, those treatment levels that exhibited approximately 50% or greater reduction in mean cell density relative to the blank control were selected for a recovery test. One recovery replicate per selected treatment level and negative control were maintained under environmental conditions similar to those in the exposure period. In order to initiate the recovery test, a 0.5 mL aliquot from each of the three replicates in the selected treatment levels were transferred to a clean flask with fresh medium. For the negative control group, a 0.5 mL aliquot was transferred from one indiscriminately selected blank control replicate to a clean flask with fresh medium. Samples of the recovery replicates were collected on days 0 and 4 of the recovery phase and were counted at the end of the recovery phase.

RANGE-FINDING STUDY
- Test concentrations: blank, 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI, 37 - 39 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI, 17 - 19 mg/L

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: none. After 72 hours of exposure, aggregation, flocculation, or adherence to the test chambers was not observed in the controls or in any treatment groups. There were no noticeable changes in cell morphology in any of the treatment groups when compared to the control replicates during the microscopic examinations of the cells.
- Recovery phase: To determine if Heloxy 107 had an algistatic or algicidal effect, a recovery test was initiated for the 20 and 64 mg formulation/L after the 72-hour exposure. In the recovery phase it was demonstrated that the effects of Heloxy 107 were algistatic (reversible) rather than algicidal, to R. subcapitata at concentrations less than or equal to 64 mg/L formulation based on greater than 16X increase of healthy cells within 72 hours.

Reported statistics and error estimates:

The calculation of cell densities, yield and growth rates and percent inhibition values, as well as all statistical analyses, were conducted using “The SAS System for Windows, Version 9.4.” The 72-hour cell density, growth rate, and yield data were evaluated for normality and homogeneity of variance (α = 0.01) using Shapiro-Wilk’s and Levene’s tests, respectively. All data met assumptions of normality and homogeneity of variance. The mean treatment group responses were compared to the negative control response using Dunnett’s one-tailed t-test (α = 0.05). ECx, ErCx, and EyCx values and their corresponding 95% confidence intervals were calculated, when possible, using non-linear regression with replicate data (cell density, growth rate, and yield, respectively) and nominal test concentrations.

Any other information on results incl. tables

Table 2, Cell densities (cells/mL)¹

Nominal concentration (mg/L)	Replicate	24 hr	48 hr	72 hr
Negative	A	68,110	575,092	3,017,156
Control	B	66,172	513,074	3,100,903
(0)	C	85,132	498,315	3,148,801
	D	76,009	452,036	2,794,402
	E	78,184	456,929	2,913,089
	F	76,057	472,572	3,117,682
0.19	A	69,880	509,826	3,141,847
	B	61,487	462,041	2,971,396
	C	64,127	489,099	3,068,164
0.61	A	72,223	531,901	3,161,909
	B	65,256	536,224	3,173,160
	C	69,143	473,344	3,080,906
2	A	61,337	494,977	2,915,896
	B	60,765	451,142	2,920,906
	C	62,738	411,327	2,761,166
6.3	A	57,608	411,982	2,604,299
	B	60,443	400,689	2,413,030
	C	55,699	379,860	2,364,937
20	A	47,199	256,778	1,384,513
	B	44,252	252,008	1,345,836
	C	41,688	236,588	1,213,695
64	A	22,270	23,851	30,976
	B	16,786	19,160	26,705
	C	16,978	24,937	29,720

1, initial cell density was 10,000 cells/mL

Table 3, growth rates (/hour)

Nominal concentration (mg/L)	Replicate	0-24 hour	24-48 hr	48-72 hr	0-72 hr
Negative	A	0.0799	0.0889	0.0691	0.0793
Control	B	0.0787	0.0853	0.0750	0.0797
(0)	C	0.0892	0.0736	0.0768	0.0799
	D	0.0845	0.0743	0.0759	0.0782
	E	0.0857	0.0736	0.0772	0.0788
	F	0.0845	0.0761	0.0786	0.0798
	CV for section growth rates	8.3%		CV for average growth rates	0.81%
0.19	A	0.0810			0.0799
	B	0.0757			0.0791
	C	0.0774			0.0795
0.61	A	0.0824			0.0799
	B	0.0782			0.0800
	C	0.0806			0.0796
2	A	0.0756			0.0788
	B	0.0752			0.0788
	C	0.0765			0.0781
6.3	A	0.0730			0.0773
	B	0.0750			0.0762
	C	0.0716			0.0759
20	A	0.0647			0.0685
	B	0.0620			0.0681
	C	0.0595			0.0667
64	A	0.0334			0.0157
	B	0.0216			0.0136
	C	0.0221			0.0151

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by a factor of > 16 (302); the mean CV for section-by-section specific growth rate in the control was < 35% (8.3%); the CV of average specific growth rates in the whole test period for control was < 7% (0.81%).

Conclusions:

The 72-hour EC50 (growth rate) of Heloxy 107 to Raphidocelis subcapitata was 38 mg/L (nominal concentration) (OECD 201). The corresponding EC10 was 18 mg/L.

Executive summary:

The 72-hour EC50 (growth rate) of Heloxy 107 to Raphidocelis subcapitata was examined in a static test conducted according to OECD 201. Test solutions were prepared as the control (0 mg/L) and at 0.19, 0.61, 2.0, 6.3, 20 and 64 mg formulation per liter. Concentrations were verified analytically for the two major components of the test material. After 72 hours, the ErC50 was reported as 38 mg/L (nominal concentration) and the ErC10 was reported as 18 mg/L (nominal concentration). The study was well-documented, followed an international standard method, and was GLP compliant; therefore, the test is considered reliable without restriction.