Zinc methacrylate

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:

27 July 2012 - 20 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.10, 0.22, 0.46 and 1.0 mg/L
- Sampling method: Quadruplicate samples were taken from the control and test media at the start (without algae) and at the end of the test (containing algae).
- Sample storage conditions before analysis: All samples were filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm) and stored deep-frozen (at about -20 °C) immediately after sampling until analysis.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of the nominal concentration of 46 mg/L was prepared by dissolving 46.01 mg of the test item completely in 1000 mL of test water using ultrasonic treatment for 10 minutes and intense stirring for 10 minutes at room temperature. The stock solution was used in a series of dilution with test water to prepare the test media.
- Controls: A control was tested in parallel containing test water without test item

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG
- Source (laboratory, culture collection): Supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Germany). The algae were cultivated at the laboratory under standardized conditions according to the test guidelines.
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): Same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

0.24 mmol/L (= 24 mg/L as CaCO3)

Test temperature:

22 °C

pH:

6.7-6.9

Dissolved oxygen:

No data

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0, 0.022, 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks containing 15 mL of test solution; continuously stirred by magnetic stirrers
- Type: Covered with a glass dish
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six
- For the 72-h stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions.

GROWTH MEDIUM
- Standard medium used: Yes; Reconstituted test water (AAP Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (AAP Medium); prepared in sterile purified water
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) was added to the test water to keep the pH of the test media as constant as possible. The pH of the test water was adjusted to 6.5 with a 1 M sodium hydroxide solution.
- Light intensity and quality: Illuminated by fluorescent tubes (Philips TLD 36W-1/840); intensity: approximately 6700 Lux (range: 6350-7350 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model Z2).
- The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, emission 680 nm). The measurements were performed at least in duplicate.
- The shape and size of the algal cells were visually inspected in the samples taken from the control and from the test concentration of nominal 0.46 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (non-concurrent)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL = 0.53-0.60; equivalent to 0.155 mg Zn/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.27 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95 % CL = 0.25-0.29

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: -

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 0.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: -

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 0.46 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Colour differences: No; all test media were clear solutions throughout the test period.
- Refer tables 6.1.5/2, 6.1.5/3 and 6.1.5/4 and figures 6.1.5/1, 6.1.5/2 and 6.1.5/3 for more details

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- Historical data (September 2012): ErC50 (72 h) = 0.93 mg/L

Reported statistics and error estimates:

- The 72-h EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95 % confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression [Davis, 1971; Finney, 1971].
- For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test [Williams, 1971; Williams, 1972] or Welch t-test [Sachs, 1984] where appropriate.

Table 6.1.5/2: Biomass of Algae

Nominal test item concentration [mg/L]	Rep. no.	Biomass of algae*
		24 h	48 h	72 h
Control	1	4.4	30.1	127.0
	2	4.5	30.0	121.8
	3	4.4	31.3	157.0
	4	3.7	25.3	124.2
	5	4.1	26.4	141.0
	6	4.0	27.4	108.3
	Mean	4.2	28.4	129.9
	SD	0.3	2.4	16.9
0.022	1	4.2	27.9	151.0
	2	4.1	26.0	121.0
	3	4.0	30.7	174.0
	Mean	4.1	28.2	148.6
	SD	0.1	2.4	26.6
0.046	1	4.2	29.6	174.9
	2	3.9	27.1	137.3
	3	4.3	29.5	120.6
	Mean	4.2	28.7	144.3
	SD	0.2	1.4	27.8
0.10	1	4.1	27.7	146.4
	2	3.9	27.0	123.5
	3	3.8	29.6	140.5
	Mean	3.9	28.1	136.8
	SD	0.1	1.3	11.9
0.22	1	3.2	18.2	83.8
	2	3.1	20.5	89.2
	3	2.9	17.5	63.9
	Mean	3.1	18.7	79.0
	SD	0.1	1.6	13.3
0.46	1	2.0	7.5	27.9
	2	2.4	9.3	32.5
	3	2.3	8.2	26.5
	Mean	2.3	8.3	28.9
	SD	0.2	0.9	3.1
1.0	1	1.0	1.2	1.6
	2	0.7	0.9	1.0
	3	0.7	1.0	1.2
	Mean	0.8	1.0	1.3
	SD	0.1	0.2	0.3

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 10³). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.93 x 10³relative fluorescence units.

Table 6.1.5/3: Average Growth Rates(µ)and inhibition of µ (Ir)

Nominal test item concentration	Average growth rate µ (day^-1) and inhibition of µ (Ir)
	0-24 h		0-48 h		0-72 h
[mg/L]	µ	Ir [%]	µ	Ir [%]	µ	Ir [%]
Control	1.51	0.0	1.71	0.0	1.64	0.0
0.022	1.48	1.5	1.71	0.2	1.69	-2.7
0.046	1.50	0.4	1.72	-0.4	1.68	-2.0
0.10	1.45	3.8	1.71	0.2	1.66	-1.1
0.22	1.20#	20.5	1.50*	12.1	1.48*	10.1
0.46	0.89#	41.0	1.09*	36.0	1.15*	30.4
1.0	-0.16#	110.5	0.04*	97.5	0.10*	93.9

*: mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller, α= 0.05)

#: mean value statistically significantly lower than in the control (according to Welch t-test, one-sided smaller, α= 0.05)

 

Table 6.1.5/4: Yield (Y) and inhibition of Y (Iy)

Nominal test item concentration [mg/L]	Yield Y (x 10^4) and inhibition of Y (Iy)
	0-24 h		0-48 h		0-72 h
	Y	Iy [%]	Y	Iy [%]	Y	Iy [%]
Control	3.3	0.0	27.5	0.0	128.9	0.0
0.022	3.2	3.1	27.3	0.7	147.7	-14.6
0.046	3.2	1.0	27.8	-1.1	143.3	-11.2
0.10	3.0	7.4	27.2	1.1	135.9	-5.4
0.22	2.1*	34.2	17.8*	35.2	78.0*	39.5
0.46	1.3*	59.1	7.4*	73.1	28.0*	78.3
1.0	-0.1*	103.9	0.1*	99.7	0.3*	99.7

*: mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller, α= 0.05)

 

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the ErC50 and EyC50 at 72 h for Zinc Dimethacrylate to green algae (Pseudokirchneriella subcapitata) were determined to be 0.56 (0.53-0.60) and 0.27 (0.25-0.29) mg/L (nominal), respectively. The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were 0.10 and 0.22 mg/L (nominal), respectively.

Executive summary:

In an algal toxicity study performed in accordance with OECD guideline 201 and GLP, Pseudokirchneriella subcapitata was exposed to Zinc Dimethacrylate at nominal concentrations of 0 (control), 0.022, 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L (three replicate flasks per test concentration and six replicates for control) for 72 h, under constant illumination and shaking in a water bath at a temperature of 22 °C. Measurement of biomass and growth inhibition was done by determination of algal biomass at 24, 48 and 72 h using microplate reader. After 72 h, cell concentration in the control culture increased by 140 folds; mean CV for section by section specific growth rate was 15 and CV for average specific growth rate was 2.6 %. Hence, it met the validity criteria for the control vessels. Chemical analysis revealed that the measured concentrations of the test item in the test media of the test concentrations 0.10-1.0 mg/L were 107-114 % of the nominal values at the start of the test and 94-108 % of the nominal values at the end of the test. Thus, the correct dosing of the test item was confirmed and it was found to be stable in the test media over the test period of 72 h. Under the test conditions, the ErC50 and EyC50 at 72 h for Zinc Dimethacrylate to green algae (Pseudokirchneriella subcapitata) were determined to be 0.56 (equivalent to 0.155 mg Zn/L) and 0.27 mg/L (nominal), respectively. The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were 0.10 and 0.22 mg/L (nominal), respectively (Eckenstein, 2013).

Description of key information

The substance exhibits an ErC50 for freshwater algae of 0.56 mg/L and an NOEC of 0.1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.56 mg/L

EC10 or NOEC for freshwater algae:

0.1 mg/L

Additional information

In an algal toxicity study performed in accordance with OECD guideline 201 and GLP, Pseudokirchneriella subcapitata was exposed to Zinc Dimethacrylate at nominal concentrations of 0 (control), 0.022, 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L (three replicate flasks per test concentration and six replicates for control) for 72 h, under constant illumination and shaking in a water bath at a temperature of 22 °C. Measurement of biomass and growth inhibition was done by determination of algal biomass at 24, 48 and 72 h using microplate reader. After 72 h, cell concentration in the control culture increased by 140 folds; mean CV for section by section specific growth rate was 15 and CV for average specific growth rate was 2.6 %. Hence, it met the validity criteria for the control vessels. Chemical analysis revealed that the measured concentrations of the test item in the test media of the test concentrations 0.10-1.0 mg/L were 107-114 % of the nominal values at the start of the test and 94-108 % of the nominal values at the end of the test. Thus, the correct dosing of the test item was confirmed and it was found to be stable in the test media over the test period of 72 h. Under the test conditions, the ErC50 and EyC50 at 72 h for Zinc Dimethacrylate to green algae (Pseudokirchneriella subcapitata) were determined to be 0.56 (equivalent to 0.155 mg Zn/L) and 0.27 mg/L (nominal), respectively. The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were 0.10 and 0.22 mg/L (nominal), respectively (Eckenstein, 2013).