Reaction mass of (2,4,6-trioxo-1,3,5-triazine-1,3,5(2H,4H,6H)-triyl)tri-2,1-ethanediyl triacrylate and 2-Propenoic acid, 1,1'-[[dihydro-5-(2-hydroxyethyl)-2,4,6-trioxo-1,3,5-triazine-1,3(2H,4H)-diyl]di-2,1-ethanediyl] ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across from short-term toxicity study on one closely structural and physicochemical related substance (TMPTA)

Justification for type of information:

The hypothesis for a read-across approach to data from THEICTA to target is the similarity of the chemical structures. Both the target substance (T) and the source substance (THEICTA) are acrylates. Study on another substance (TMPTA, S2) is used as supporting study. Both the target substance (T) and TMPTA (S2) are triacrylates. The main difference is the core group to which the acrylates are attached. Both core groups are shown to be non-toxic and will not contribute to the observed toxicity.
For further read-across see document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Conclusions:

Effects of THEICTA on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) were determined: EC10 = 12.9 mg/L (95% confidence level: 7.1 - 23.5 mg/L); EC50 = 25.7 mg/L (95% confidence level: 12.7 - 51.3 mg/L)

TMPTA (S2) tested for aquatic toxicity against the algae Scenedesmus subspicatus according to the method DIN 38412/9. After 72h exposure the aquatic toxicity was determined to be: ErC10 = 1.9 mg/l and ErC50= 18.8 mg/l

The hypothesis for a read-across approach to data from THEICTA to target is the similarity of the chemical structures. Both the target substance (T) and the source substance (THEICTA) are acrylates. Study on another substance (TMPTA, S2) is used as supporting study. Both the target substance (T) and TMPTA (S2) are triacrylates. The main difference is the core group to which the acrylates are attached. Both core groups are shown to be non-toxic and will not contribute to the observed toxicity.
For further read-across see document attached in section 13.

Executive summary:

Effects of THEICTA on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) were determined: EC10 = 12.9 mg/L (95% confidence level: 7.1 - 23.5 mg/L); EC50 = 25.7 mg/L (95% confidence level: 12.7 - 51.3 mg/L)

TMPTA (S2) tested for aquatic toxicity against the algae Scenedesmus subspicatus according to the method DIN 38412/9. After 72h exposure the aquatic toxicity was determined to be: ErC10 = 1.9 mg/l and ErC50= 18.8 mg/l

The hypothesis for a read-across approach to data from THEICTA to target is the similarity of the chemical structures. Both the target substance (T) and the source substance (THEICTA) are acrylates. Study on another substance (TMPTA, S2) is used as supporting study. Both the target substance (T)  and TMPTA (S2) are triacrylates. The main difference is the core group to which the acrylates are attached. Both core groups are shown to be non-toxic and will not contribute to the observed toxicity.

For further read-across see document attached in section 13.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-16 to

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 were taken from all test concentrations and the control at the start of the test and after 72 hours of exposure. Inoculated samples were previously filtered through 0.45µm then analysed.

Vehicle:

no

Details on test solutions:

Tests solutions were prepared from dilutions of a stock solution: the test item density is 1.3, 76.9 µL of test item previously heated up to 60°C (corresponding to 100 mg) was thus pipetted and introduced in a 500 mL flasks capacity to get the target addition rate for this test of 200 mg/L of test item. The volume was then made up with the test medium. The solution was kept under high speed stirring at ambient temperature during approximately 20 hours with a magnetic stir bar. The range of concentrations was obtained from successive dilutions.

Algae were exposed to the following series of nominal concentrations: 0.9, 1.9, 4.2, 9.4, 20.6, 45.4 and 100.0 mg/L. An inoculated control flask was prepared and incubated under the same conditions, with no test item. Three vessels were prepared at each test concentration and six vessels for the control group; each vessel was inoculated with ca 0.5 x 10^4 cells/mL P. subcapitata.

Test organisms (species):

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

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata, Strain No. CCAP 278/4, supplied by the Culture Centre of Algae and Protozoa (Ambleside, UK).

Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline.

The quality of the stock culture was checked for the absence of micro-organisms and deformed or abnormal cells under microscopic observation before use.

Four days before the start of the exposure, two pre-cultures were prepared by inoculating sufficient cells from the algal stock culture to the growth medium to obtain a low cell density (5.10^3 cells/mL) for pre-culturing, in order to maintain exponential growth until the start of the test. The pre-cultures were incubated under the same conditions as those used for the test cultures. Only one of the two pre-cultures was used to inoculate the test flasks for the study. The second one was only to be used if the first one was damaged.

At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to obtain an initial cell concentration of 5.10^3 as recommended in the OECD 201 guideline.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.2 to 22.6 °C

pH:

t0: 7.9
t72: 7.9 - 8.0

Dissolved oxygen:

t0: 8.1 - 8.2 mg/L
t72: 8.6 - 8.8 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0 (Control), 0.9, 1.9, 4.2, 9.4, 20.6, 45.4, 100.0 mg/L

Details on test conditions:

The incubation was conducted in a phytoculture cabinet that allows test flasks to be incubated under precise conditions: temperature was set to 23 ± 1°C; flasks were continuously shaken with a rotation speed at around 100 rpm and constantly illuminated by fluorescent tubes between 6,000 and 10,000 lux.

Test vessels were made of glass bottles (120 mL capacity) capped with cellulose bungs. They were filled with a volume of 50 mL.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Reference item test

The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate. The latest value of ErC50 obtained in May 2017 was 1.11 mg/L.

According to the ISO 8692 standard, the 72h-ErC50 obtained from an inter-laboratory exercise on potassium dichromate was in the range 0.65 to 1.73 mg/L range.

Algae test

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, no precipitation was observed at the end of the test.

 

Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm.

Chemical analysis of test samples taken at 0 and 72h indicated that measured concentrations of the test item remained within 80-120% of the nominal concentrations and were maintained over the 72h test period (i.e. deviation <20%), except at the 1.9 mg test item/L concentration for which the test item was not detected (an analytical deficiency was suspected). This concentration was discarded for the statistical analysis: the nominal concentrations 0.9, 4.2, 9.4, 20.6, 45.4 and 100.0 mg/L were used for the statistical analysis.

 

Cell densities, biomass, growth rate and inhibition percentages are summarised in the tables below:

Test solution	Replicate	Algal density at 24h (Fd, ¢ *10^4/ ml )			Algal density at 48h (Fd, ¢ *10^4/ ml )			Algal density at 72h (Fd, ¢ *10^4/ ml )			Average specific growth rate J0-J3				Specific growth rate inhibition (%)
		Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Average	Sx	CV
Control	a	2.27	1.99	10.35	8.70	7.17	13.00	41.70	46.39	16.49	1.475	1.506	0.055	3.674	Inhibition (per replicate)	Inhibition (mean)
	b	2.08			7.11			36.15			1.427
	c	2.11			7.69			57.32			1.581
	d	1.76			6.28			51.89			1.547
	e	1.96			7.06			48.32			1.524
	f	1.75			6.21			42.92			1.484
0.90	a	2.49	2.28	7.97	10.31	9.17	10.89	57.50	58.04	17.79	1.582	1.581	0.060	3.769	-5.0	-5.0
	b	2.14			8.77			47.99			1.521				-1.0
	c	2.21			8.44			68.62			1.641				-8.9
1.90	a	2.30	2.05	10.47	8.79	8.00	8.65	47.18	46.18	12.33	1.516	1.507	0.042	2.787	-0.6	0.0
	b	1.91			7.66			40.05			1.461				3.0
	c	1.95			7.53			51.31			1.544				-2.5
4.20	a	2.21	2.24	1.85	8.71	8.50	5.84	44.47	47.70	30.35	1.496	1.509	0.099	6.591	0.7	-0.2
	b	2.23			7.94			35.11			1.417				5.9
	c	2.29			8.87			63.53			1.615				-7.2
9.40	a	2.03	2.08	3.44	6.64	6.63	4.37	29.43	29.70	26.93	1.358	1.353	0.092	6.775	9.8	10.2
	b	2.06			6.34			21.84			1.259				16.4
	c	2.17			6.92			37.84			1.442				4.3
20.60	a	1.43	1.44	3.35	3.65	3.72	4.76	14.20	10.93	42.61	1.115	1.002	0.171	17.084	25.9	33.5
	b	1.39			3.60			5.60			0.805				46.5
	c	1.49			3.93			12.98			1.086				27.9
45.40	a	0.67	0.66	1.63	1.37	1.34	6.50	2.10	1.40	62.27	0.479	0.276	0.290	104.830	68.2	81.7
	b	0.65			1.24			1.69			0.406				73.1
	c	0.65			1.41			0.42			-0.055				103.7
100.00	a	0.57	0.57	4.53	0.42	0.43	1.94	0.09	0.08	10.16	-0.569	-0.598	0.035	-5.770	137.8	139.7
	b	0.59			0.43			0.07			-0.636				142.2
	c	0.54			0.44			0.09			-0.590				139.2

Test solution (mg/L)	Growth rate Iµi (%)	Yield Iyi (%)
0.9	-5.0	-25.4
1.9	0.0	0.5
4.2	-0.2	-2.9
9.4	10.2	36.4
20.6	33.5	77.3
45.4	81.7	98.0
100.0	139.7	100.9

The results (growth rate) demonstrated that algal growth was inhibited from -5.0% at the lowest concentration (i.e. 0.9 mg/L nominal concentration) to >100% at the highest tested concentration (100.0 mg/L).

Dissolved oxygen and pH are presented in the table below. Measurements were carried out in non-inoculated solution at T₀and inoculated solution at T₇₂.An increase in the pH is observed, this may be associated to consumption of the dissolved CO₂due to the growth of algae.

Measured pH and dissolved oxygen

Test solution (mg/L)	pH		Dissolved O2 (mg/L)
	0h	72h	0h	72h
Control	7.9	8.0	8.1	8.6
0.9	7.9	8.0	8.1	8.8
1.9	7.9	8.0	8.1	8.7
4.2	7.9	8.0	8.1	8.7
9.4	7.9	7.9	8.1	8.7
20.6	7.9	7.9	8.1	8.7
45.4	7.9	7.9	8.1	8.6
100.0	7.9	7.9	8.2	8.6

NA: Not Applicable

 

Validation criteria

The test met the validity criteria of the test guideline detailed as follows:

 

§ The biomass in the control cultures increased exponentially by a factor of 92.8 within the 72-hour test period (required: at last 16-fold)

 

§ The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were less than or equal to 21.1% (required: < 35%)

 

§ The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 3.7% (required: < 7%)

 

 

Validity criteria fulfilled:

yes

Conclusions:

The results are reported as growth rate and yield inhibitions. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined:

Growth rate:
EC10 = 12.9 mg/L (95% confidence level: 7.1 - 23.5 mg/L)
EC50 = 25.7 mg/L (95% confidence level: 12.7 - 51.3 mg/L)

Yield:
EC10 = 4.6 mg/L (95% confidence level: 2.3 - 9.2 mg/L)
EC50 = 11.3 mg/L (95% confidence level: 5.1 - 24.7 mg/L)

Executive summary:

This study was designed to determine the effects of the test item on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline.

 

Tests solutions were prepared from dilutions of a stock solution, algae were exposed to the following series of nominal concentrations: 0.9, 1.9, 4.2, 9.4, 20.6, 45.4 and 100.0 mg/L.

 

As there was evidence that the concentration of the substance being tested were satisfactorily maintained within ± 20% of the nominal or measured initial concentration throughout the test, analysis of the results was based on nominal values (the 1.9 mg/L concentration was discarded for the final calculations).

 

The results are reported as growth rate and yield inhibitions. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined:

Growth rate:

EC10 = 12.9 mg/L (95% confidence level: 7.1 - 23.5 mg/L)

EC50 = 25.7 mg/L (95% confidence level: 12.7 - 51.3 mg/L)

Yield:

EC10 = 4.6 mg/L (95% confidence level: 2.3 - 9.2 mg/L)

EC50 = 11.3 mg/L (95% confidence level: 5.1 - 24.7 mg/L)

The test met the validity criteria of the test guideline detailed as follows:

 

§ The biomass in the control cultures increased exponentially by a factor of 92.8 within the 72-hour test period (required: at last 16-fold)

 

§ The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were less than or equal to 21.1% (required: < 35%)

 

§ The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 3.7% (required: < 7%)

 

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-10-02 - 1989-10-6

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

4.86 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Cell density

Concentration		0h	24h	48h	72h	96h
[mg/l]		N/ml x 10^3	N/ml x 10^3	N/ml x 10^3	N/ml x 10^3	N/ml x 10^3
control		12	26	98	301	547
1		12	26	83	221	407
2.5		12	26	74	212	375
5		12	26	64	180	300
10		12	24	54	128	189
25		12	20	36	60	64
50		12	17	21	21	17

Evaluation after 72h

Concentration	Growth rate	Biomass inhibition [%]	Rate-related inhibition [%]
[mg/l]
control	1.07	--	--
1	0.97	20.4	9.6
2.5	0.96	27.0	10.9
5	0.90	37.5	16.0
10	0.79	51.5	26.5
25	0.54	73.1	50.1
50	0.19	88.5	82.6

Evaluation after 96h

Concentration	Growth rate	Biomass inhibition [%]	Rate-related inhibition [%]
[mg/l]
control	0.95	--	--
1	0.88	25.6	7.7
2.5	0.86	30.1	9.9
5	0.80	41.7	15.7
10	0.69	59.6	27.8
25	0.42	82.7	56.2
50	0.09	95.1	90.9

after 72 h:

EBC10:       0.605 mg/l                     EuC10: 1.9 mg/l

EBC50:        7.157 mg/l                    EuC50: 18.849 mg/l

after 96 h:

EBC10:       0.573 mg/l                   EuC10: 2.184 mg/l

EBC50:        4.859 mg/l                 EuC50: 14.535 mg/l

Validity criteria fulfilled:

not specified

Conclusions:

After 72h exposure the aquatic toxicity was determined to be: ErC10 = 1.9 mg/l and ErC50= 18.8 mg/l

Executive summary:

The test substance was tested for aquatic toxicity against the algae Scenedesmus subspicatus according to the method DIN 38412/9. After 72h exposure the aquatic toxicity was determined to be: ErC10 = 1.9 mg/l and ErC50= 18.8 mg/l

Description of key information

The toxicity to algae of the target substance is based on a read-across to THEICTA. Studies on TMPTA is used as supporting study.

THEICTA

The toxicity of THEICTA (part of the target substance) to algae was determint to be ErC10=12.9 mg/L and ErC50=25.7 mg/L.

TMPTA

The toxicity of TMPTA (S2) to algae was determined to be: ErC10 = 1.9 mg/l and ErC50= 18.8 mg/l

The hypothesis for a read-across approach to data from THEICTA to target is the similarity of the chemical structures. Both the target substance (T) and the source substance (THEICTA) are acrylates. Study on another substance (TMPTA, S2) is used as supporting study. Both the target substance (T)  and TMPTA (S2) are triacrylates. The main difference is the core group to which the acrylates are attached. Both core groups are shown to be non-toxic and will not contribute to the observed toxicity. For further read-across see document attached in section 13.

Key value for chemical safety assessment

EC50 for freshwater algae:

25.7 mg/L

EC10 or NOEC for freshwater algae:

12.9 mg/L

Additional information

The test substance was tested for aquatic toxicity against the algae Pseudokirchneriella subcapitata according to the method OECD 201.

The study is reliable and was assigned as key study.

After 72h exposure the aquatic toxicity was determined to be: EC10(growth rate) = 12.9 mg/l, EC50(growth rate) = 25.7 mg/l.