N-[3-(dimethylamino)propyl]methacrylamide

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:

2008-07-14 - 2008-07-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sample storage conditions before analysis: Room temperature, protected from moisture and light

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen

- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE•m-2•s-1 for 24 h per day.
- Culturing media: Nutrient medium Z according to LÜTTGE et al. (1994) :

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

4 - 12 d

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

Nominal range: 21 - 24 +/- 2 °C

Nominal and measured concentrations:

nominal: 11.5, 28.8, 72.0, 180 450 mg/l
The concentrations of the active ingredient Dimethylaminopropyl methacrylamide were analysed at all concentration levels at test start and test end via LC-MS. The measured concentrations at test start were in the range of 92 - 118 % of the nominal values. At the end of the test the test item gave recoveries of 89 – 101 %. All effect values are given based on nominal test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs
- Initial cells density: A three day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with dilution water. For the start of the test the preculture was directly pipetted to the test and control replicates to receive an initial cell concentration of approximately 2 – 5 x 103 cells/mL in the replicates.

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
see table

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: see table
- Photoperiod: 24 h/d light
- Light intensity and quality: 60 - 120 µE • m-2 • s-1

EFFECT PARAMETERS MEASURED
Recovery of algae: After 72 h algae were transferred from test concentrations
72.0 – 450 mg/L and control into fresh untreated medium
(0.5 – 1 mL algae suspension from each replicate were filled up with dilution water to 10 mL) and allowed to grow for further
4 - 12 d to determine whether the effect of the item is reversible.

TYPE AND FREQUENCY OF MEASUREMENT: Cell density was measured via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. The cell density was measured at the beginning of the test (after application) and every 24 h. Dilution water was used as background signal. The pH-value at the beginning of the test was measured out of one additional replicate of each test concentration and control. At the end it was measured from one pool per concentration of all replicates. The room temperature was measured continuously by a hygrothermograph. Light intensity was measured prior to test start. Microscopic evaluation of the cells at the start and at the end of the incubation was determined. Cells were checked for any unusual cell shapes, colour differences, chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of alga cells.

COURSE OF THE STUDY:
Based on the results of a preliminary range finding test (non GLP), a definitive test was performed with 5 concentration levels in a geometrical series with a factor 2.5 and 3 replicates each. A control with dilution water (without test item, six replicates) was tested under the same conditions as the test group. A three day-old preculture incubated at study conditions was used for the main study. Chlorophyll-fluorescence was determined at the beginning and after 24, 48 and 72 h. Recovery of algae was carried out as specified above. Other measurements were performed as specified above.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study
- Test concentrations: 10, 100 and 500 mg/l (nominal)
- Results used to determine the conditions for the definitive study: 500 mg/l 100 % Specific growth rate inhibition, 100 % Specific yield inhibition
100 mg/l: 24 % Specific growth rate inhibition, 71 % Specific yield inhibition

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

45.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 43.4 - 47.4 mg/l

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

53.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 51.1 - 56.1 mg/l

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

70.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 67.1 - 73.6 mg/l

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

68.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 67.3 - 70.2 mg/l

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

77.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 75.6 - 78.4 mg/l

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

94.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 92.3 - 95.6 mg/l

Details on results:

After 72 h algae were transferred from nominal concentrations 72.0 - 450 mg/L and control to fresh untreated medium and allowed to grow for further 4 - 12 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 450 mg/L (highest test concentration).

Results with reference substance (positive control):

The toxicity of potassium dichromate to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 h from March 10 to 13, 2008

EC50 Values of the Reference Item based on nominal concentrations mg/L, (0-72 h):

Yield inhibition
EyC50 0.31
95 % confidence interval 0.30 – 0.33
Rate-related inhibition
ErC50 0.74
95 % confidence interval 0.70 – 0.78

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures according to CLOPPER and PEARSON (1934).
see attached document

EC-Values (0-72 h) and 95 % Confidence Intervals ofDimethylaminopropyl methacrylamide

based on nominal concentrations of the test item [mg/L]

	Inhibition of yield
EyC10	45.4 (43.4 – 47.4)
EyC20	53.3 (51.1 – 56.1)
EyC50	70.5 (67.1 – 73.6)
	Rate-related inhibition
ErC10	68.9 (67.3 – 70.2)
ErC20	77.3 (75.6 – 78.4)
ErC50	94.2 (92.3 – 95.6)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
450		1		1928		0	0	1140
		2		1928		0	0	760
		3		1928		0	0	760
		Mean		1928		0	0	887
180		1		1928		1612	1014	1140
		2		1928		2285	1427	1520
		3		1928		1550	394	760
		Mean		1928		1816	945	1140
72.0		1		1928		7568	56948	266416
		2		1928		8651	55721	261737
		3		1928		8120	66533	261454
		Mean		1928		8113	59734	263202
28.8		1		1928		12551	112721	572992
		2		1928		14150	126037	535238
		3		1928		14855	104600	539607
		Mean		1928		13852	114453	549279
11.5		1		1928		17831	115412	570768
		2		1928		13586	108084	489280
		3		1928		13530	126527	588061
		Mean		1928		14982	116674	549370
Control		1		1928		12583	93071	501528
		2		1928		17958	98921	535424
		3		1928		14840	113040	497159
		4		1928		14690	106865	598881
		5		1928		14144	133699	565402
		6		1928		12814	118081	628579
		Mean		1928		14505	110613	554496

Evaluation after 72 h

Nominal test item concentration	Replicate	Yield		Inhibition of yield	Growth rate		Rate-related inhibition
[mg/L]	No.			[%]	[d^-1]		[%]
450	1		-788	100.00		-0.175	100.00
	2		-1168	100.00		-0.310	100.00
	3		-1168	100.00		-0.310	100.00
	Mean		-1041	100.00		-0.265	100.00
180	1		-788	100.00		-0.175	100.00
	2		-408	100.00		-0.079	100.00
	3		-1168	100.00		-0.310	100.00
	Mean		-788	100.00		-0.188	100.00
72.0	1		264488	52.13		1.643	12.89
	2		259809	52.98		1.637	13.20
	3		259526	53.03		1.637	13.22
	Mean		261274	52.71		1.639	13.10
28.8	1		571064	-3.35		1.898	-0.65
	2		533310	3.49		1.875	0.56
	3		537679	2.69		1.878	0.41
	Mean		547351	0.94		1.884	0.11
11.5	1		568840	-2.94		1.897	-0.58
	2		487352	11.80		1.845	2.14
	3		586133	-6.07		1.907	-1.11
	Mean		547442	0.93		1.883	0.15
Control	1		499600			1.854
	2		533496			1.876
	3		495231			1.851
	4		596953			1.913
	5		563474			1.894
	6		626651			1.929
	Mean		552568			1.886

Section-by-Section and Average Specific Growth Rates of the Control Group (0 – 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	± SD	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	1.876	2.001	1.684	1.854	0.160	8.61	14.5
	2	2.232	1.706	1.689	1.876	0.309	16.5
	3	2.041	2.030	1.481	1.851	0.320	17.3
	4	2.031	1.984	1.723	1.913	0.166	8.66
	5	1.993	2.246	1.442	1.894	0.411	21.7
	6	1.894	2.221	1.672	1.929	0.276	14.3
				Mean	1.886
				SD ±	0.03
				CV [%]	1.68

SD = Standard deviation    CV = percent coefficient of variation

 

  Recovery of Alga Cells

After 72 h of exposure, 0.5 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from nominal test item concentrations 72.0 - 450 mg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 4 - 12 days under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 450mg/L.

Nominal test item concentration [mg/L]	Incubation time                   [d]	Cell density [cells/mL]		Growth rate after incubation time [d^-1]
		Initial^#	Final
450	12	89	636	0.16
			624	0.16
			831	0.19
180	6	114	718654	1.46
			638343	1.44
			537265	1.41
72.0	4	13160	1739808	1.22
			1743720	1.22
			1651839	1.21
Control		27725	1466497	0.99
			1624581	1.02

^#) calculated from the cell density of the definitive test after 72 h

Validity criteria fulfilled:

yes

Remarks:

The study meets the validity criteria of the guideline: cell growth, test temperature, difference temperature from mean, pH value in the control, mean coefficients of variation of average spec. growth rates were fulfilled.

Conclusions:

In this study Dimethylaminopropyl methacrylamide was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of yield (EyC50) and specific growth rate (ErC50) after 72 h were 70.5 (67.1 – 73.6) and 94.2 (92.3 – 95.6) mg/L, respectively.
The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 4 - 12 days of growth under test conditions.
All effect levels are given based on nominal concentrations of Dimethylaminopropyl methacrylamide.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Desmodesmus subspicatus were exposed to Dimethylaminopropyl methacrylamide at nominal concentrations of 11.5, 28.8, 72.0, 180 and 450 mg/L under static conditions in accordance with OECD 201. The EC10, EC20 and EC₅₀values based on growth rate were measured.

After 72 h algae were transferred from nominal concentrations 72.0 - 450 mg/L and control to fresh untreated medium and allowed to grow for further 4 - 12 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 450 mg/L (highest test concentration).

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for Alga, Growth inhibition test with Desmodesmus subspicatus toxicity study.

 

Results Synopsis

Test Organism: Desmodesmus subspicatus

Test Type: Static 

72 hr ErC50:  94.2 mg/l (95% Confidence interval: 92.3 - 95.6 mg/l)  

72 hr ErC10:  68.9 mg/l (95% Confidence interval: 67.3 - 70.2 mg/l)  

  

            

Description of key information

The ErC50/ ErC10 value based on growth rate was 94.2/ 68.9 mg/L and EbC50 value based on yield was 70.5 mg/L after 72 hours,

respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

94.2 mg/L

EC10 or NOEC for freshwater algae:

68.9 mg/L

Additional information

There is one ecotoxicity study in freshwater algae available for Dimethylaminoproyl methacrylamide, which is considered reliable without restriction (Klimisch score 1; DR.U.NOACK-LABORATORIEN, 2009).

In an 72 hour acute toxicity study (static dose-response test), the cultures of Desmodesmus subspicatus strain no. CHODAT SAG 86.81 were exposed to Dimethylaminopropyl methacrylamide at nominal concentrations of 0, 11.5, 28.8, 72.0, 180, and 450 mg/L under static conditions in accordance with the guideline OECD 201. The ErC₁₀value based on growth rate was 68.9 mg/L after 72 hours, respectively