3,6,9-trioxaundecamethylene dimethacrylate

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:

2017-09-25 to 2018-01-15

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution into the test chambers and prior to inoculation. At exposure termination, samples were collected from the pooled replicates from each treatment and control group. At each sampling interval, 20 mL of test solution was collected from mid-depth and transferred into glass scintillation vials. At each sampling interval, an additional set of samples was collected and stored under refrigerated conditions for possible future analysis.

Vehicle:

no

Details on test solutions:

A primary stock was prepared by dissolving 0.1054 g of the test substance in 1000 mL of freshwater AAP medium to achieve a nominal concentration of 100 mg a.i./L. The primary stock was stirred for fourteen minutes prior to use and continued stirring while all subsequent dilutions were made. The primary stock
appeared clear and colorless. Four additional test solutions were prepared at nominal concentrations of 6.3, 13, 25, and 50 mg a.i./L by diluting aliquots of the 100 mg a.i./L primary stock with freshwater AAP medium. The 6.3, 13, 25, and 50 mg a.i./L test solutions were mixed by inversion and appeared clear and colorless and
were otherwise unremarkable at the time of preparation. The negative control solution consisted of freshwater AAP medium without test substance added.

Test organisms (species):

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

Details on test organisms:

The freshwater alga, Raphidocelis subcapitata, was selected as the test species for this study. The species is representative of an important group of freshwater algae, and was selected for use in the test based upon a past history of use, and ease of culturing in the laboratory. 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. Algal cells used in this test were obtained from EAG Laboratories – Easton cultures that had been actively growing in culture medium under similar environmental conditions as used in
this test for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium four days prior to test initiation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24.02 - 24.21 °C (Table 3, see "Any other information on results incl. tables")

pH:

7.4 - 9.4 (Table 4, see "Any other information on results incl. tables")

Nominal and measured concentrations:

Nominal concentrations: 0, 6.3, 13, 25, 50, 100 mg a.i./L
Mean measured concentrations: 0, 5.8, 12, 24, 49, 99 mg a.i./L

Table 2, see "Any other information on results incl. tables"

Details on test conditions:

Test chambers were held in an environmental chamber at a temperature of 24 ± 2ºC. The temperature of a container of water adjacent to the test chambers in the environmental chamber was measured continuously using an Amega Scientific Corporation centralized monitoring system. The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was 6,000 lux ± 10%. Light intensity was measured at test solution level at five locations surrounding the test flasks at test initiation using a SPER Scientific 840006C light meter. The pH of the medium in each treatment and control group was measured at test initiation and exposure termination using a Thermo Orion A214 pH meter, or equivalent. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and control group. At exposure termination, pH was measured in pooled samples of test solution collected from each of the replicates of each treatment and control group.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

37 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

68 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

RESULTS AND DISCUSSION

Measurement of Test Concentrations

Results of analyses to measure concentrations of tetraethylene glycol dimethacrylate in test solution samples collected during the study are presented in Table 2. Measured concentrations of tetraethylene glycol dimethacrylate on day 0 in the treatment groups ranged from 99.1 to 102% of nominal. Measured concentrations of tetraethylene glycol dimethacrylate at test termination in the treatment groups ranged from 81.1 to 97.3% of nominal. Mean measured concentrations were determined to be 5.8, 12, 24, 49, and 99 mg a.i/L, equivalent to 92.7, 90.0, 96.8, 98.0, and 98.7% of nominal, respectively.

Table 2 Measured Concentrations of Tetraethylene Glycol Dimethacrylat ein Freshwater AAP Medium

 

Nominal Test Concentration (mg a.i./L)		Sample Number (524P-102-)	Sampling Time (Days)	Measured Concentration (mg a.i./L)^1,2	Percent of Nominal^2	Mean Measured Concentration (mg a.i./L)	Mean Measured Percent of Nominal
0.0		1	0	< LOQ	--	--	--
		7	3	< LOQ	--	--	--
6.3		2	0	6.45	102	5.8	92.7
		8	3	5.22	82.8
13		3	0	12.9	99.1	12	90.0
		9	3	10.5	81.1
25		4	0	25.4	101	24	96.8
		10	3	22.9	91.5
50		5	0	50.5	101	49	98.0
		11	3	47.5	95.1
100		6	0	100	100	99	98.7
		12	3	97.3	97.3
	^1  The limit of quantitation (LOQ)was 2.50 mg a.i./L, defined as the lowest nominal concentration in a fortified sample with an acceptable recovery of 70-110%.  ^2   Results were generated using Agilent OpenLab CDS Rev A.01.05. Manual calculations may differ slightly.

Observations and Measurements

Measurements of temperature, pH, and light intensity are presented in Tables 3, 4, and 5, respectively. Temperatures remained within the 24 ± 2°C range established for the test. The pH of all test solutions at test initiation ranged from 7.4 to 7.5. At test termination, pH measurements in samples of test solution obtained by pooling the replicates of each experimental group ranged from 7.7 to 9.4. The pH increased by more than 1.5 units over the course of the study, however, the photosynthetic activity of the test organism increases pH

over time. The increase in pH was not considered to detrimental to the study since growth in the control replicates achieved all validity criteria. The light intensity ranged from 5,780 to 6,450 lux, which was within the desired range of 6,000 lux ± 10%.

Table 3 Temperature Measurements

Time (Days)	Temperature (°C)^1
	Average	Low	High
0	24.17	24.02	24.21
1	24.20	24.15	24.21
2	24.18	24.15	24.21
3	24.14	24.08	24.15
0-3	24.17	24.02	24.21
^1 Temperature was continuously monitored in a container of water located adjacent to the study with an Amegaview Scientific centralized monitoring system.

 

 

Table 4 pH Measurements

Mean Measured Test Concentration (mg a.i./L)	pH Measurements
	Day 0^1	Day 3^2
Negative Control	7.5	9.2
5.8	7.4	9.4
12	7.4	9.4
24	7.4	9.1
49	7.4	8.0
99	7.4	7.7
^1    Day 0 samples were collected from the individual batches of test solution prepared for each treatment and control group at test initiation. ^2    Day 3 samples were from pooled samples of test solution collected from the three replicates per treatment and six replicates in the control group at test termination.

 

Table 5 Light Intensity Measurements on Day 0

 

Shaker Table ID#	Light Intensity (lux)^1
	Measurement No. 1	Measurement No. 2	Measurement No. 3	Measurement No. 4	Measurement No. 5	Mean ± S.D.
AQL#14	5,780	6,190	6,430	6,150	6,450	6,200 ± 271.3
^1  Measurements taken at five locations surrounding the test flasks at test solution level.

The toxicity of tetraethylene glycol dimethacrylate to R.subcapitata was determined by evaluating changes in cell density over a 72-hour exposure period. Cell densities were used to calculate growth rates for each 24-hour interval of exposure and yields at 72 hours of exposure. Mean growth rates, cell densities, and yields and their corresponding percent inhibition values after 72 hours of exposure are presented in Table 6. NOEC, ECx ErCx, and EyCx values and their corresponding 95% confidence limits for cell densities, growth

rates, and yield, respectively, are presented in Table 7. The 24 and 48-hour EC50 values for cell density and growth rate are presented in Appendix 6. Individual replicate data for each growth parameter are presented in Appendices 7 and 8. Exponential growth, characterized by the linear section of the growth curve, occurred throughout the duration of the test in the negative control replicates.

Table 6

 

Inhibition ofR. subcapitataGrowth After 72 Hours of Exposure toTetraethylene Glycol Dimethacrylate

 

72 Hours of Exposure^1
Mean Measured Test Concentration (mg a.i./L)		Mean Cell Density (cells/mL)^3	Percent Inhibition^2		Mean Growth Rate (hours^-1)	Percent Inhibition^2		Mean Yield (cells/mL)^3	Percent Inhibition^2
Negative Control		3,250,000	--		0.0803	--		3,240,000	--
5.8		2,780,000*	14		0.0781	3		2,770,000*	15
12		2,623,333*	19		0.0774	4		2,613,333*	19
24		2,080,000*	36		0.0740*	8		2,070,000*	36
49		753,333*	77		0.0599*	25		743,333*	77
99		35,000*	99		0.0172*	79		25,000*	99
^1 Calculations were performed using SAS Version 9.4. Manual calculations may differ slightly. ^2 Inhibition was calculated relative to the mean negative control response. ^3 Yield was calculated as the final cell density minus the nominal initial cell density (10,000 cells/mL). * Treatment group means were significantly different from the negative control mean (Dunnett’s test,p < 0.05).

Table 7

 

NOEC, ECx, E_rCx, and E_yCx Values After 72 Hours of Exposure

 

Endpoint		Based on Mean Measured Concentrations
Growth Rate:
ErC50^1		68 mg a.i./L
95% Confidence Interval		66 to 71 mg a.i./L
ErC10^1		37 mg a.i./L
95% Confidence Interval		34 to 41 mg a.i./L
NOEC^2		12mg a.i./L
Cell Density:
EC50^1		32 mg a.i./L
95% Confidence Interval		28 to 37 mg a.i./L
EC10^1		16 mg a.i./L
95% Confidence Interval		12 to 20 mg a.i./L
EC5^3		13 mg a.i./L
95% Confidence Interval		9.9 to 17 mg a.i./L
NOEC^2		< 5.8mg a.i./L
Yield:
EyC50^1		32 mg a.i./L
95% Confidence Interval		28 to 37 mg a.i./L
EyC10^1		16 mg a.i./L
95% Confidence Interval		13 to 21 mg a.i./L
EC5^3		13 mg a.i./L
95% Confidence Interval		10 to 18 mg a.i./L
NOEC^2		< 5.8mg a.i./L
^1  ErCx, ECx, and EyCxvalues were estimated, when possible, using non-linear regression with treatment response and mean measured concentrations.  ^2  NOEC values were determined based on the results of statistical comparisons between treatment groups and the negative control. ^3  An experimental NOEC could not be determined with the data obtained.  EC05value was determined and reported.

Appendix 6

 

EC₅₀and E_rC₅₀Values After 24 and 48 Hours of Exposure Based on Mean Measured Concentrations

 

Endpoint		24 Hours	48 Hours
Growth Rate:
ErC50^1		79 mg a.i./L	62 mg a.i./L
95% Confidence Interval		67 to 95 mg a.i./L	58 to 67 mg a.i./L
Cell Density:
EC50^1		68 mg a.i./L	41 mg a.i./L
95% Confidence Interval		50 to 92 mg a.i./L	34 to 49 mg a.i./L
^1  ErCxandECxvalues were estimated, when possible, using non-linear regression with treatment response and mean measured test concentrations.

Appendix 7 Cell Density and Yield by Replicate Over the 72-Hour Exposure Period

 

Mean Measured Concentration (mg a.i./L)	Replicate	Cell Density (cells/mL)^1,2						Yield (cells/mL)^3
		24 Hours		48 Hours		72 Hours		72 Hours
Negative Control	A	59,000	620,000		2,760,000		2,750,000
(0.0)	B	43,000	420,000		3,270,000		3,260,000
	C	71,000	445,000		3,390,000		3,380,000
	D	45,000	690,000		3,530,000		3,520,000
	E	57,000	395,000		3,330,000		3,320,000
	F	62,000	450,000		3,220,000		3,210,000
5.8	A	73,000	490,000		2,850,000		2,840,000
	B	66,000	470,000		3,000,000		2,990,000
	C	49,000	360,000		2,490,000		2,480,000
12	A	62,000	490,000		2,660,000		2,650,000
	B	55,000	485,000		2,580,000		2,570,000
	C	57,000	465,000		2,630,000		2,620,000
24	A	32,000	510,000		2,440,000		2,430,000
	B	50,000	545,000		1,760,000		1,750,000
	C	51,000	330,000		2,040,000		2,030,000
49	A	54,000	163,000		670,000		660,000
	B	32,000	154,000		900,000		890,000
	C	36,000	159,000		690,000		680,000
99	A	18,000	15,000		28,000		18,000
	B	17,000	22,000		33,000		23,000
	C	22,000	14,000		44,000		34,000
^1The initial cell density of the stock culture was determined and an inoculum volume was administered to each test chamber to yield a cell density of approximately 10,000 cells/mL at test initiation (0 hours). ^2  Mean cell density in the negative control replicates after 72 hours was 3,250,000 cells/mL, exceeding the 16X growth requirement. ^3  Yield was calculated as the final cell density in the exposure period minus the initial cell density.

Appendix 8 Growth Rate by Replicate Over the 72-Hour Exposure Period

 

Mean Measured Concentration (mg a.i./L)	Replicate	Growth Rate (per hour)
		0 - 24 Hours	0 – 48 Hours	0 – 72 Hours
Negative Control	A	0.0740	0.0860	0.0781
(0.0)	B	0.0608	0.0779	0.0804
	C	0.0817	0.0791	0.0809
	D	0.0627	0.0882	0.0815
	E	0.0725	0.0766	0.0807
	F	0.0760	0.0793	0.0802
5.8	A	0.0828	0.0811	0.0785
	B	0.0786	0.0802	0.0792
	C	0.0662	0.0747	0.0766
12	A	0.0760	0.0811	0.0775
	B	0.0710	0.0809	0.0771
	C	0.0725	0.0800	0.0774
24	A	0.0485	0.0819	0.0764
	B	0.0671	0.0833	0.0718
	C	0.0679	0.0728	0.0739
49	A	0.0703	0.0581	0.0584
	B	0.0485	0.0570	0.0625
	C	0.0534	0.0576	0.0588
99	A	0.0245	0.0084	0.0143
	B	0.0221	0.0164	0.0166
	C	0.0329	0.0070	0.0206

After 72 hours of exposure, inhibition of cell density in the 5.8, 12, 24, 49, and 99 mg a.i./L treatment groups (based on mean measured concentrations) was 14, 19, 36, 77, and 99%, respectively, relative to the negative control. Inhibition of growth rate in the 5.8, 12, 24, 49, and 99 mg a.i./L treatment groups was 3, 4, 8, 25, and 79%, respectively, relative to the negative control. Inhibition of yield in the 5.8, 12, 24, 49, and 99 mg a.i./L treatment groups was 15, 19, 36, 77, and 99%, respectively, relative to the negative control (Table 6).

Dunnett’s test indicated mean cell density and mean yield were significantly reduced (p < 0.05) in all tetraethylene glycol dimethacrylate treatment groups when compared to the negative control. Dunnetts’s test indicated that mean growth rate was significantly reduced (p < 0.05) in the 24, 49, and 99 mg a.i./L treatment groups when compared to the negative control. The 72-hour NOEC was determined to be <5.8 mg a.i./L, based on statistically significant reductions in cell density and yield. EC05 and EyC05 values and their corresponding 95% confidence intervals were estimated since an experimental NOEC was not determined. The 72-hour EC05 and EyC05 values were determined to be 13 mg a.i./L, for both endpoints.

After 72 hours of exposure, adherence of cells to the test chambers was not observed in any of the control or treatment groups. Flocculation or aggregation of cells was not observed in any of the experimental groups. Cells in the 99 mg a.i./L treatment group appeared enlarged when compared to cells in the negative control. Cells present in all other treatment groups appeared normal when compared to cells in the negative control.

Conditions for the Validity of the Test

Mean cell density in the control flasks increased by a factor of 325 after three days, achieving the 16X growth criterion. The coefficient of variation of average specific growth rates in the control replicates was 1.5%, achieving the less than 7% criterion. The mean percent coefficient of variation for section-by-section specific growth rates in the control replicates was 16.6%, achieving the less than 35% criterion (Appendix 9).

Appendix 9 Specific Growth Rate of the Negative Control

 

Section-by-Section Growth Rate for Negative Control

 

Control Replicate	0-24 Hour Growth Rate	24-48 Hour Growth Rate	48-72 Hour  Growth Rate	% Coefficient of Variation^1
A	0.0740	0.0980	0.0622	23.4
B	0.0608	0.0950	0.0855	22.0
C	0.0817	0.0765	0.0846	5.09
D	0.0627	0.1138	0.0680	34.5
E	0.0725	0.0807	0.0888	10.1
F	0.0760	0.0826	0.0820	4.53
			Mean:	16.6
^1Coefficient of Variation = S.D./mean *100 Results were generated using Excel 2010 in the full precision mode. Manual calculations may differ slightly.

 

 

Average Specific Growth Rate for the Negative Control

 

Control Replicate	0-72 Hour Growth Rate
A	0.0781
B	0.0804
C	0.0809
D	0.0815
E	0.0807
F	0.0802
Mean= Standard Deviation= % COV^1=	0.0803 0.0012 1.5
^1Coefficient of Variation = S.D./mean *100 Results were generated using Excel 2010 in the full precision mode. Manual calculations may differ slightly.

Validity criteria fulfilled:

yes

Conclusions:

The freshwater alga, Raphidocelis subcapitata, was exposed to a series of five treatment levels of tetraethylene glycol dimethacrylate ranging from 5.8 to 99 mg a.i./L, based on mean measured concentrations. The toxicity of tetraethylene glycol dimethacrylate to Raphidocelis subcapitata was assessed based on effects on cell density, growth rate, and yield relative to the negative control. The 72-hour NOEC was determined to be <5.8 mg a.i./L, based on statistically significant reductions in mean cell density and mean yield in all tetraethylene glycol dimethacrylate treatment groups after 72 hours of exposure. EC05 and EyC05 values and their corresponding 95% confidence intervals were estimated since an experimental NOEC was not determined. The 72-hour EC05 and EyC05 values were determined to be 13 mg a.i./L, for both endpoints. The 72-hour EC10 values for cell density, growth rate, and yield were determined to be 16, 37, and 16 mg a.i./L, respectively. The 72-hour EC50 values for cell density, growth rate, and yield were determined to be 32, 68, and 32 mg a.i./L, respectively.

Executive summary:

The freshwater alga, Raphidocelis subcapitata, was exposed to a series of five treatment levels of tetraethylene glycol dimethacrylate ranging from 5.8 to 99 mg a.i./L, based on mean measured concentrations. The toxicity of tetraethylene glycol dimethacrylate to Raphidocelis subcapitata was assessed based on effects on cell density, growth rate, and yield relative to the negative control. The 72-hour NOEC was determined to be <5.8 mg a.i./L, based on statistically significant reductions in mean cell density and mean yield in all tetraethylene glycol dimethacrylate treatment groups after 72 hours of exposure. EC05 and EyC05 values and their corresponding 95% confidence intervals were estimated since an experimental NOEC was not determined. The 72-hour EC05 and EyC05 values were determined to be 13 mg a.i./L, for both endpoints. The 72-hour EC10 values for cell density, growth rate, and yield were determined to be 16, 37, and 16 mg a.i./L, respectively. The 72-hour EC50 values for cell density, growth rate, and yield were determined to be 32, 68, and 32 mg a.i./L, respectively.

Description of key information

A study assessing the toxicity of the test item to Pseudokirchneriella subcapitata was conducted in accordance with the OECD 201 Test Guideline and GLP requirements.

The 72h-EC50 based on growth rate inhibition was determined to be 68 mg/L and the EC10 based on the same parameter was determined to be 37 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

68 mg/L

EC10 or NOEC for freshwater algae:

37 mg/L