7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

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:

01 Sep - 08 Jul 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

pH changes exceeded the 1.5 units during the test, temperature was higher than defined in the guideline.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations were sampled and measured at the beginning and at the end of the test

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the test item is not sufficient soluble, thus the water accommodated fractions (WAF) were prepared in a second analytical test performed for 0 and 100 mg test item/L. The nominal load was weighed, adding the corresponding amount of dilution water and shaking vigorously for 24 h. The solution was then filtered (45 µm filters) and used to prepare the treatments.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Chodat
- Source: collection of alga, Institut für Pflanzenphysiologie, University of Goettingen, Germany
- Age of inoculum (at test initiation): 5 d
- Method of cultivation: algae are kept as stock culture on solid agar at 8 °C

ACCLIMATION
- Acclimation period: 96 h
- Culturing media and conditions: an aliquot of the stock culture containing a few cells was transferred into pre-culture medium. This culture was then diluted 1:1 with pre-culture medium and incubated for another 24 h. The resulting culture was growing exponentially. After the adjustment to a cell concentration of about 6 x 10^4 cells/mL through photometric measurements and addition of algal medium, the culture was usable for the test. The adjusted pre-culture was mixed with the same amount of 10-fold-nutrient solution. This mixture was the culture used for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

28 - 32 °C

pH:

7.6 - 9.2 (control), 7.6 - 7.8 (test item)

Nominal and measured concentrations:

0 (control), 4.6, 10, 22, 46 and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: round cuvettes d = 10 mm
- Fill volume: 5 mL (was stirred during incubation)
- Type (delete if not applicable): open
- Initial cells density: 6 x 10^4 cells/mL (verified photometrically)
- Control end cells density: 1454575 cells/mL
- No. of vessels per concentration (replicates): 3 at the main test, 6 at the limit test
- No. of vessels per control (replicates): 6 for both main and limit test

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Pre-culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test

OTHER TEST CONDITIONS
- Light intensity and quality: 6100 - 6200 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: by photometric measurement of optical density at the beginning of the incubation and every 24 h

TEST CONCENTRATIONS
- Limit test: in a first experiment conducted as a limit test with 100 mg test item/L (pH: 7.6 - 8.6, temperature: 29 - 31 °C), an inhibition of 15% based on growth rate and 48.11% based on yield was observed after 72 h. Based on these results five concentrations ranging from 4.6 - 100 mg/L were tested in the main test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: corresponding to 100 mg/L nominal concentration

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: corresponding to 100 mg/L nominal concentration

Details on results:

- Exponential growth in the control (for algal test): yes, increase by a factor of 117 in the main study

Results with reference substance (positive control):

- Results with reference substance valid: yes
- ErC50 (72 h): 0.82 mg/L (95% CI 0.68 - 1.12 mg/L)
- NOEC (72 h): 0.16 mg/L

Reported statistics and error estimates:

For the treatment with the test item concentrations of 0.21 and 0.68 mg/L (46 and 100 mg/L nominal), it was evaluated with a t-test whether the differences between treatment and control were significant.

Biological results base on the mean measured concentrations.

Table 1: Measured concentrations in mg/L and the corresponding recovery raters in %.

Nominal concentr. in mg/L	Measured concentr. in mg/L		Recovery after 72 h	% of nominal concentr.		Mean of measured concentr. in mg/L
	0 h	72 h		0 h	72 h
0	< LOQ	< LOQ	-	-	-	-
4.6	< LOQ	< LOQ	-	-	-	< LOQ
10	0.17	< LOQ	-	1.7	-	-
22	< LOQ	< LOQ	-		-	< LOQ
46	0.44	0.1	23%	1.0	0.2	0.21
100	1.28	0.36	28%	1.3	0.4	0.68

Low recoveries of the test item at the end of the study (after 72 h) could be attributed to adsorption of the test material on the algal cells. Because of the unexpected low measured concentrations an additional experiment was performed with water accommodated fractions of the test item and the results are shown in table 2.

Table 2: Measured concentrations of the test item when prepared as WAF in both deionised and dilution water filtered and unfiltered.

Nominal concentr. in mg/L	Measured concentr. in mg/L in deionised water		Measured concentr. in mg/L in dilution water
	filtered	unfiltered	filtered	unfiltered
100	4.9	5.1	2	1.8

Table 3: Growth rate and yield as well as % of inhibition values in the main study.

Nominal concentr. in mg/L	Mean growth rate/day	% Inhibition growth rate	Mean yield [cell concentr./mL]	% Inhibition yield
0	1.59	0	1442125	0
4.6	1.59	-0.09	1448350	-0.43
10	1.58	0.33	1419300	1.58
22	1.59	-0.41	1471175	-2.01
46	1.60	-0.81	1500225	-4.03
100	1.55	2.31	1290650	10.5

For the treatment with the test item concentrations of 0.21 and 0.68 mg/L (46 and 100 mg/L nominal), it was evaluated with a t-test whether the differences of growth rate as well as yield between treatment and control were significant.

Table 4: Significance evaluation via t-test.

Nominal concentr. in mg/L	46 mg/L		100 mg/L
Endpoint	Growth rate	Yield	Growth rate	Yield
Calculated t-value	2	2	11.62	11.21
Tabulated t-value	2	2	2.36	2.36
Significance	no	no	yes	yes

Table 5: Further effect concentrations of the tested item.

NOEC (yield)	0.21 mg/L
LOEC (yield)	0.68 mg/L
EbC50	> 0.68 mg/L
EC50 (yield)	> 0.68 mg/L
EC100 (growth rate)	> 0.68 mg/L
EC100 (biomass)	> 0.68 mg/L
EC100 (yield)	> 0.68 mg/L

Validity criteria fulfilled:

yes

Description of key information

ErC50 (72 h) > 0.68 mg/L (mean measured, corresponding to 100 mg/L nominal) for Desmodesmus subspicatus based on growth rate (OECD 201)

NOErC (72 h) = 0.21 mg/L (mean measured) for Desmodesmus subspicatus based on growth rate (OECD 201)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.21 mg/L

Additional information

One study is available, in which the toxicity of the substance (CAS 72869-86-4) to algae was investigated. Two experiments are listed in the report. In the first one a limit test with 100 mg/L was conducted and due to observed inhibition effects a second study with 5 concentrations was set up. The water accommodated fractions (WAF) were prepared. The nominal load was weighed, adding the corresponding amount of dilution water and shaking vigorously for 24 h. The solution was then filtered (45 µm filters) and used to prepare the treatments. The test was conducted according to OECD 201 and GLP. The freshwater green algae Desmodesmus subspicatus was exposed in the main study to the nominal test item concentrations of 0 (control), 4.6, 10, 22, 46 and 100 mg/L for 72 h. An analytical monitoring was performed via HPLC-UV-VIS resulting to low recoveries of the tested substance (max 28%). Biological effects were observed and resulted in NOEC (72 h) of 0.21 mg/L and EC50 (72 h) of > 0.68 mg/L (both mean measured, based on growth rate).