Tricarbonyl(methylcyclopentadienyl)manganese

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:

29-01-2010 to 07-04-2010

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:

yes

Remarks:

See explanation below

Principles of method if other than guideline:

Deviations: 1. In one of the 6 replicates of the solvent-control (combined limit/range-finding test) no algae were added. Evaluation: The remaining 5 replicates with algae were sufficient for assessment of the results of the test. 2. The pH of some test solutions exceeded 9.0 at the end of the 48-hour test period (combined limit/range-findind test and final test). Evaluation: the maximum measured pH was 9.3, which only just exceeds the optimum pH range. The increase was caused by using closed systems and could not be prevented despite the fact that extra bicarbonate was used in the media and the pH of the test media was adjusted to pH 7.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.46, 1.0, 2.2, 4.6 and 10 mg/l
- Sampling method: singular samples for analysis were taken from all test concentrations and the solvent-control.
- Frequency: t=0h, t=24h and t=48h
- Sample storage conditions before analysis: not applicable, samples were analysed on the day of sampling

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of test solutions started with stock solutions in acetone at a factor of 10,000 higher than the final test concentrations. Volumes of 100 µl of the respective stocks were added per litre of test medium and stirred one day in a closed vessel with minimal headspace. The final test solutions were all clear and colourless.
- Differential loading: 1 x 10^4 cells/ml
- Controls: blank control (test medium without test substance or other additive) and solvent control
- Chemical name of vehicle : acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100µl/l
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: none
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture
- Age of inoculum (at test initiation): 3 or 4 days
- Method of cultivation: cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

ACCLIMATION
- Acclimation period: not relevant, in-house culture
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No.

Hardness:

24 mg CaCO3/L.

Test temperature:

Between 22.6 and 23.1 °C

pH:

Between 6.0-9.0

Dissolved oxygen:

Not relevant.

Salinity:

Not relevant.

Nominal and measured concentrations:

See table 1.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume:glass, 100 ml capacity filled with 50 ml test solution, 50% headspace
- Aeration: yes
- Type of flow-through : n.a. static
- Initial cells density:10^4 cells/ml
- Control end cells density: solvent-control - 33.9 x 10^4 cells/ml; blank-control - 32.8 x 10^4 cells/ml
- No. of organisms per vessel: 50 x 10^4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: M2; based on OECD 201 guideline but with a double amount of NaHCO3. In addition, pH was adjusted to 7.
- Detailed composition if non-standard medium was used: not relevant

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water (tap water purified by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, to 7
- Photoperiod: ilumination continously
- Light intensity and quality: TLD lamps of the type 'cool-white' of 30 Watt, with a light intensity within the range of 87 to 94 µE.m^-2.s^-1
- Salinity (for marine algae): not relevant

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cells densities were determined by spectophotometric measurement of samples at 720 nm using a Varian Cary single beam spectrophotometer with a cuvette (pathlenght = 10 mm). Algal medium was used as blank
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: n.a.
- Range finding study
- Test concentrations: 0.1, 1.0 and 10 mg/l
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate at concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 1.2-2.3 mg/l

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.29-0.58 mg/l

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.07 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate reduction and yield inhibition

Remarks on result:

other: -

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.16-0.28 mg/l

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.08-0.17 mg/l

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): cells had a normal appearance
- Unusual cell shape: no
- Colour differences: no
- Flocculation: none
- Aggregation of algal cells:
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no, highest concentration tested (10 mg/l) was within solubility range

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 for growth rate reduction: 1.2 mg/l 95% C.I. 0.77-2.0 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hen ce the EC50 for the present batch corresponds with this range.
- EC50 for yield inhibition: 0.44 mg/l 95% C.I. 0.37-0.54 mg/l. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/l. Hence the EC50 for the present batch corresponds with this range.

Reported statistics and error estimates:

The NOEC and the EC50 were calclated per recommendations in the OECD guideline 201. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-test, TOXSTAT Release 3.5). Additionally, the EC10 was determined to meet the recommendations asset forthe in “A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines” (August 1993. Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.

Table 2 – Mean cell densities (x10⁴cells/ml) during the final test

Concentration mmt TWA (mg/l)	Exposure time
	0	24	48
Solvent-control	1.0	6.7	33.9
Blank-control	1.0	5.7	32.8
0.07	1.0	6.2	30.3
0.14	1.0	5.4	29.4
0.30	1.0	3.9	19.7
0.61	1.0	3.9	12.3
1.3	1.0	2.0	6.5

Table 3 – Percentage reduction of the growth rate (total test period) and percentage inhibition of yield during the final test

Concentration mmt TWA (mg/l)	Mean growth rate		Yield (0 - 48 h)
	µ (0-48 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
Solvent-control	0.07939		32.86
Blank-control	0.07253	0.9	31.81	3.2
0.07	0.07588	3.1	29.34	10.7
0.14	0.07039	4.0	28.39	13.6
0.30	0.06204	15.4	18.72	43.0
0.61	0.05231	28.7	11.33	65.5
1.3	0.03909	46.7	5.53	83.2

 

Table 4 – Percentage reduction of growth rate at different time intervals during the final test

Concentration mmt TWA (mg/l)	Mean growth rate		Yield (0 - 48 h)
	µ (0-24 h)	Reduction (%)	µ (24-48 h)	Reduction (%)
Solvent-control	0.07939		0.06730
Blank-control	0.07253	8.6	0.07286	-8.3
0.07	0.07588	4.4	0.06619	1.6
0.14	0.07039	11.3	0.07045	-4.7
0.30	0.05511	30.6	0.06898	-2.5
0.61	0.05549	30.1	0.04914	27.0
1.3	0.02796	64.8	0.05023	25.4

 

Table 5 – pH levels recorded during the final test

Concentration mmt TWA (mg/l)	Exposure time (hours
	0	48
Solvent-control	7.6	9.2
Blank-control	7.7	9.2
0.07	7.4	9.3
0.14	7.5	9.3
0.30	7.5	8.8
0.61	7.5	8.3
1.3	7.5	8.1

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, mmt reduced the growth rate and inhibited the yield of this fresh water algae species significantly at TWA concentrations of 0.14 mg/l and higher.
The EC50 for growth rate reduction (ERC50: 0-48h) was 1.7 mg/l with a 95% confidence interval ranging from 1.2 to 2.3 mg/l.
The EC50 for yield inhibition (EyC50: 0-48h) was 0.41 mg/l with a 95% confidence interval ranging from 0.29 to 0.58 mg/l.
The NOEC for both growth rate reduction and yield inhibition were determined to be a TWA concentration of 0.07 mg/l.

Executive summary:

The study was performed per OECD 201 with minor deviations. The procedures were designed to meet the specifications of EC/440/2008, Part C.3, 2008; Amended by EC/761/2009, ISO International Standard 8692, 2004 and the OECD series on Testing and Assessment Number 23, 2000.

The mmt tested was a clear yellow-orange liquid with a purity >96%. The test material was completely soluble in the test medium at the concentrations tested.

Stock solutions in acetone at a factor of 10,000 higher than the final test concentrations were used for spiking. A subsequent stirring period of one day resulted in a completely dissolved clear and colourless test solutions.

A final test was performed based on the result of a preceding combined limit/range-finding test. Exponentially growing algal cultures were exposed to a blank and solvent-control and to nominal mmt concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg/l. The test design was optimized for testing a suspected volatile substance using completely closed test vessels and a reduced test period of 48 hours. The initial algal cell density was 10⁴cells/ml and samples for analytical confirmation of actual exposure concentrations were taken at the start of the test period and after both 24 and 48 hours of exposure.

Analyses of the samples showed that measured concentrations were in agreement with nominal at the study start (93-98% relative to nominal), however, the measured concentrations decreased to 6 -7% of nominal during the first 24-hour test period and further to <2% of nominal after 48-hours. The range tested based on TWA concentrations corresponded with 0.07, 0.14, 0.30, 0.61 and 1.3 mg/l. The analytical results of the highest test concentration incubated without algae showed a slightly better stability in time indicating that aside from the expected volatility and light sensitivity, adsorption may have played a (minor) role in the observed decrease in test concentration.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

mmt reduced growth rate and inhibited the yield of this fresh water algae species significantly at TWA concentrations of 0.14 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-48h) was 1.7 mg/l with a 95% confidence interval ranging from 1.2 to 2.3 mg/l.

The EC50 for yield inhibition (EyC50: 0-48h) was 0.41 mg/l with a 95% confidence interval ranging from 0.29 to 0.58 mg/l.

The NOEC for both growth rate reduction and yield inhibition equaled a TWA concentration of 0.07 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.7 mg/L

EC10 or NOEC for freshwater algae:

0.07 mg/L