Molybdenum zinc tetraoxide

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:

11 October 2018 - 10 January 2019

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration of the test item was measured at each test concentration respectively the control at the beginning and end of the test in triple determination. For analytical determinations a separate set of test vessels (cuvettes, PS, with lids) were prepared. Additionally two test solutions without algae were prepared for the analytical determination.

Vehicle:

no

Details on test solutions:

A stock solution with 36 mg/l was prepared by dissolving the test item in growth medium. The solution was be stirred overnight at room temperature afterwards. The next day the stock solution was filtered, so that only the dissolved proportion was tested. The test solutions were prepared by adding growth medium and inoculum culture to the stock solution of the test item. The samples were filtered (0.45 μm) to remove the algae, preserved with acid (HNO3, 65 %) and cool storage.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Source (laboratory, culture collection): Laboratory
- Method of cultivation: In order to adapt the test algae to the test conditions and ensure that the algae are in the exponential growth phase when used to inoculate the test solutions, an preparatory culture in the growth medium was prepared 4 days before start of the test.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: The algae showed a normal and healthy appearance under the microscope after the incubation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.9 - 23.3 °C.

pH:

7.60-8.72

Nominal and measured concentrations:

Nominal concentrations: 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L
Measured concentration (initial):
Measured concentration (final):

Details on test conditions:

TEST SYSTEM
- Test vessel: Cuvettes (PS)
- Type (delete if not applicable): Closed (lids)
- Material, size, headspace, fill volume: 3 mL
- Aeration: No.
- Initial cells density: 3.0*10E3 cell/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: Uniform fluorescent illumination with an average light intensity of 8430 lux
- Other: Constantly shaken.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algal biomass in each test vessel was measured as chlorophyll fluorescence daily during the test period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0.09, 0.9, 9, 90 and 900 mg/L (72 hours, static)
- Results used to determine the conditions for the definitive study: -3, 5, 94, 109 and 71% of growth rate inhibition respectively. The lower inhibition of the highest concentration versus the lower concentration could be a result of a stressinduced growth in the first 24 hours. The expected ErC50-value will be between 1 and 9 mg/l.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No.
- Unusual cell shape: No.
- Colour differences: No.
- Flocculation: Unknown.
- Adherence to test vessels: Unknown.
- Aggregation of algal cells: No.
- Any stimulation of growth found in any treatment: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The recovery of the test item was between 25 and 38 % at the beginning of the test. In the test solutions with nominal concentrations < 1.0 mg/l the measured concentration were slightly below the limit of quantification. It appears that the test item reacts with the growth medium and has formed a precipitate that was not visible to the naked eye. Adsorption effects or filtering then apparently removed part of the test item from the test solution. At the end of the test the measurements of the nominal concentrations greater than 2.0 mg/l show that the test item was stable over the test period. The measured concentration of the nominal concentration of 1.0 mg/l was not slightly, but well below the limit of quantification at the end of the test. This measurement suggest that there was absorption or adsorption effects over the test period. One possible explanation is that there was adsorption effects of the test item e. g. by the algae. As a result, part of the test item could have been removed from the sample by filtration prior to analysis. This could have led to the different concentrations between the beginning and the end of the test at the nominal concentration of 1.0 mg/l.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

The reliability of the test conditions was tested with the reference item (positive control) potassium dichromate (K2Cr2O7) in a separate study (R18-1001-010) at 18./21.09.2018. The determined ErC50 of 0.73 mg/l lies within the range from the inter laboratory test to ISO 8692 (0.84 ± 0.24 mg/l). So the test conditions were reliable.

Reported statistics and error estimates:

From the determined effects the EC50-, EC20- and EC10-values of the test item were calculated by non-linear regression. In addition, the NOEC/LOEC of the test item were calculated from the average specific growth rate and the yield. In both cases the calculation was carried out with the software SigmaPlot 13.

Nominal and determined concentrations:

Nominal concentration of ZnMoO4 (mg/L)	Correspondingdetermined concentration of ZnMoO4		Average of the measured concentration of Zn
	at 0 hours (mg/L)	at 72 hours (mg/L)	at 0 hours (mg/L)	at 72 hours (mg/L)
0 (control)	<0.327	<0.327	<0.095	<0.095
0.25	<0.327	<0.327	<0.095	<0.095
0.5	<0.327	<0.327	<0.095	<0.095
1.0	<0.327 (0.298) (2)	<0.327	<0.095 (0.087) (2)	<0.095
2.0	0.714	0.624	0.207	0.181
4.0	0.993	0.845	0.288	0.245
8.0	2.82	2.88	0.817	0.834
0 (blank) (1)	<0.327	<0.327	<0.095	<0.095
0.25 (1)	<0.327	<0.327	<0.095	<0.095
8.0 (1)	3.04	2.79	0.883	0.811

(1) test solutions without algae

(2) calculation based on measured/ determined concentration slightly below the limit of quantification

Recovery:

Nominal concentration ofZnMoO4 (mg/L)	Recoveryof ZnMoO4
	at 0 hours (%)	at 72 hours (%)
0.25	-	-
0.5	-	-
1.0	29.8 (2)	-
2.0	35.7	31.2
4.0	24.8	21.1
8.0	35.2	35.9
0.25 (1)	-	-
8.0 (1)	38.0	34.9

The recovery of the test item was between 25 and 38 % at the beginning of the testI. Thus, it can be assumed that the test item was stable over the test duration. Because the recovery was lower than 80 %, the results below were calculated with the analytical concentrations at the beginning of the test. The results of the nominal concentration of 1.0 mg/l were also used to calculate the effect concentrations, because the analytical concentration were only slightly lower than the limit of quantification. The results of the nominal concentration of 0.25 and 0.5 mg/l were not taken into account when calculating the effect concentrations.

Percent inhibition of the growth rate:

ZnMoO4Conc. (mg/L)	Parallel	Inhibition of the growth rate (%)		Inhibition of the yield (%)
		Single value	Mean	Single value	Mean
<0.33 (0.25) (1)	a	0.2	1.0	0.9	4.2
	b	0.7		3.3
	c	1.9		8.3
<0.33 (0.5) (1)	a	5.0	5.8	20.1	22.5
	b	4.8		19.3
	c	7.4		28.2
0.30 (2)	a	8.2	7.0	30.7	26.7
	b	5.8		22.9
	c	7.0		26.6
0.71	a	12.6	11.0	42.9	38.6
	b	9.8		35.3
	c	10.6		37.7
0.99	a	23.1	20.9	64.4	60.6
	b	19.8		58.7
	c	19.8		58.7
2.82	a	69.6	70.2	96.5	96.6
	b	71.5		96.8
	c	69.4		96.4

(1) test solutions without algae

(2) calculation based on measured/ determined concentration slightly below the limit of quantification

Calculated effect concentrations after 72 hours:

	Average specific growth rate			Yield
	Concentration (mg/L)	95% Confidence Limits		Concentration (mg/L)	95% Confidence Limits
		lower	upper		lower	upper
EC10	0.47	0.33	0.61	<0.33	-	-
EC20	1.1	0.98	1.2	<0.33	-	-
EC50	2.2	2.1	2.2	0.84	0.77	0.93
NOEC	<0.33	-	-	<0.33	-	-
LOEC	0.33	-	-	0.33	-	-

Growth rates [μ/ day] of the control cultures:

Control	Test period (0-1 d)		Test period (1-2 d)		Test period (2-3 d)		Test period (0-2d)		Test period (0-3d)
	[µ / d]		[µ / d]		[µ / d]		[µ / d]		[µ / d]
	Single value	Mean	Single value	Mean	Single value	Mean	Single value	Mean	Single value	Mean
a	1.527	1.324	1.381	1.627	1.430	1.442	1.453	1.477	1.445	1.465
b	1.477		1.467		1.494		1.472		1.479
c	1.357		1.623		1.460		1.492		1.481
d	1.201		1.805		1.367		1.506		1.459
e	1.250		1.673		1.425		1.464		1.451
f	1.132		1.811		1.477		1.475		1.476

Coefficient of variation (CV) in the control cultures:

Test period [d]	CV [%]
0-1	11.9
1-2	10.8
2-3	3.1
Mean of the daily CV	8.6
Whole test duration (0-3)	1.1

The growth curve of the control group shows that the controls have largely grown exponentially over the course of the test. This can be seen from the almost straight course of the curve with logarithmic application of the biomass (here as fluorescence). The growth of the controls in the last 24 hours of the test duration was not substantial reduced. The control cultures achieved over the whole test duration an average growth rate of 1.5 d-1. This corresponds to an increasing biomass by a factor of 80. The mean coefficient of variation of the daily growth rates was 8.6 %, the coefficient of variation over the whole test period was 1.1 %.

Validity criteria fulfilled:

yes

Remarks:

(biomass in control cultures increased by a factor >16; mean coefficient of variation for section-by-section specific growth rate in control cultures <35%; coefficient of variation of average specific growth rates in replicate control cultures < 7%)

Conclusions:

The ErC50 and ErC10 were determined to be 2.2 mg/L and 0.84 mg/L respectively after 72 hours in green algae.

Executive summary:

The growth inhibition test of Desmodesmus subspicatus was performed according to the OECD Guideline 201 (GLP study). Based on preliminary results, 3.0*10E3 cell/ml for replicate were exposed to 0 (control), 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L test item (3 replicates each) for 72 hours unders static conditions. The algal biomass in each test vessel for the biological determination was measured as chlorophyll fluorescence daily during the test period. The algal growth was determined from the measurement of the algal biomass. The average specific growth rate and the yield and the percentage of inhibition of the growth rate and yield were calculated for each test replicate and the controls for the different periods. For analytical determinations of test item concentrations a separate set of test vessels (cuvettes, PS, with lids) were prepared. Additionally two test solutions without algae were prepared for the analytical determination. The recovery of the test item was between 25 and 38 % at the beginning of the test but the substance was found to be stable. Because the recovery was lower than 80 %, the results were calculated with the analytical concentrations at the beginning of the test. All validity criteria were fulfilled. The calculation of the average specific growth rates and the yield of the treatment replicates occurred with the measured biomass without removing of outliers. The 72h-ErC50 and 72h-EyC50 were determined to be 2.2 mg/L and 0.84 mg/L respectively. The 72h-ErC10 and 72h-EyC10 were determined to be 0.47 mg/L and <0.33 mgL respectively. The NOEC resulted to be lower than 0.33 mg/L.

Description of key information

Toxicity to aquatic algae and cyanobacteria: Key study: Test method OECD 201. GLP study. The ErC50 and ErC10 were determined to be 2.2 mg/L and 0.47 mg/L respectively after 72 hours in green algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.2 mg/L

EC10 or NOEC for freshwater algae:

0.47 mg/L

Additional information

According to the OECD Guideline 201, recent scientific developments have led to a recommendation of abandoning the concept of NOEC and replacing it with regression based point estimates ECx. An appropriate value for x has not been established for this algal test. A range of 10 to 20 % appears to be appropriate (depending on the response variable chosen), and preferably both the EC10 and EC20 should be reported. Therefore, for chronic effects the ErC10 value has been taken into account.