Dipotassium propanedioate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.01-09.02.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium propanedioate

EC Number:

820-064-0

Cas Number:

13095-67-5

Molecular formula:

C3H2K2O4

IUPAC Name:

Dipotassium propanedioate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analytical samples were taken at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged test solutions from all test item concentration and control. For each sampling also a retain sample was taken.

Test solutions

Vehicle:

no

Details on test solutions:

- AAP-Medium (according to Annex 3 of OECD 201)
- The pH was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary.

Test organisms

Test organisms (species):

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

Details on test organisms:

The algae are grown semi-continuously in sterile cultures in the laboratory.
Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state.
Stock cultures are ordered regularly from our commercial supplier.
Cells from this semi-continuous liquid stock culture were used for the test.
3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

determination of cell division after 24, 48 and 72 hours of exposure

Post exposure observation period:

none

Test conditions

Hardness:

not specified

Test temperature:

22.2 – 22.8 °C

pH:

7.20 – 8.08 (control)

Dissolved oxygen:

not specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

Nominal concentration: 100, 31.3, 9.77, 3.05 and 0.954 mg/L.
Measured initial concentrations ranged from 91 % to 107% of nominal.
Measured final concentrations were below LOQ.

Details on test conditions:

TEST SYSTEM
- Initial cells density: 0.5 × 104 cells/mL
- Number of replicates: Six replicates were employed for the control and three for each test item concentration

TEST MEDIUM
- AAP-Medium

TEST CONDITIONS
- Test procedure: Dose response test (static)
- Duration: 72 hours
- Temperature: 22.2 – 22.8 °C
- CO2 supplied: By continuous agitation
- pH of control: 7.20 – 8.08
- Illumination: Continuously, 88.8 µEm-2s-1 (mean)
- Photoperiod: continuous illumination


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): yield and growth rate
- Determination of number of cells: fluorescence measurement.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: 100, 31.3, 9.77, 3.05, 0.954 mg/L and control.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 5.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Details on results:

- Validity Criteria of the Study:
-- Biomass: Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 95.59, which exceeds the threshold of 16. It corresponds to a growth rate of 1.52043 d-1
-- Coefficient of Variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 25 % and did not exceed 35 %
-- Coefficient of Variation (average growth rate): The coefficient of variation of average growth rate in replicate control cultures was 2.1 % and did not exceed 7 % for the whole test period

- Biological Results:
After 72 h at termination of the test no inhibition of growth rate or yield was observed for test item concentrations up to and including 31.3 mg/L. At the highest test item concentration of 100 mg/L the inhibition of growth rate peaked in 13.3 % and the inhibition of yield peaked in 45.5 % at a nominal test item concentration of 100 mg/L.
The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item concentration of 100 mg/L. At 100 mg/L nominal test item concentration fewer cells were observed than at the lower concentrations.
The growth conditions (pH and temperature) during the test were within the range specified by OECD 201.
The mean light intensity of all positions of the incubator was 88.8 µEm-2s-1 with mean values of each platform in a range from 82.6 to 93.6 µEm-2s-1 (-7.0 % to +5.4 % of mean) which was within ± 15 % of variation as specified by OECD 201.

- Analytical Results:
The measured initial concentrations of dipotassium malonate ranged from 91 % to 107% of nominal. In the aged samples the measured concentrations were between

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010).
A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test.
The NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided, for growth rate and yield.
The calculation of the EC10, 20, 50 was not indicated due to a weak concentration response relation and since the inhibition was below 50 % at the highest test item concentration for both, yield and growth rate and hence the database was inappropriate for probit analysis.

Any other information on results incl. tables

Toxicological endpoints for the test item

	Test item [mg/L]
	nominal	actual^3)
ErC10(Growth rate)^1)	n.d.	n.d.
ErC20^1)	> 100	> 5.75
ErC50^1)	> 100	> 5.75
EyC10(Yield)^1)	n.d.	n.d.
EyC20^1)	n.d.	n.d.
EyC50^1)	> 100	> 5.75
NOEC^2)	31.3	2.06
LOEC^2)	100	5.75

¹⁾Due to an inhibition below 50% the database was weak for probit analysis which hence was not performed

²⁾Following Dunnetts-t-test (left-sided, p<0.05)for growth rate and for yield

³⁾Based on geometric mean of each concentration level

n.d.: Not determined

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 100 mg/L (nominal) and 5.75 mg/L (actual).
The overall LOEC was therefore determined to be 100 mg/L (nominal) and 5.75 mg/L (actual), the corresponding NOEC was set at 31.3 mg/L (nominal) and 2.06 mg/L (actual).
The EC20- and EC50-value for growth rate and the EC50-value for yield were considered to be > 100 mg/L (nominal) and 5.75 mg/L (actual).

Executive summary:

The objective of this study was to determine the effects of Dipotassium malonate on the growth of the single cell green alga Pseudokirchneriella subcapitata, to determine the no observed effect concentration (NOEC), to determine the lowest observed effect concentration (LOEC) and to determine the effect concentration (EC_{10, 20, 50}), where possible. The study was performed according to OECD TG 201 and in compliance to GLP.

Initial target cell densities of 0.5 × 10⁴ cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours.

Where possible inhibition of growth was assessed by the determination of NOEC/LOEC and EC_{10, 20, 50} for growth rate and yield after 72 hours.

A static test with nominal test item concentrations of 100, 31.3, 9.77, 3.05, 0.954 mg/L and control was performed.

Six replicates were employed for the control and three for each test item concentration.

Analytical samples were taken and analysed from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged test solutions.

NOEC and LOEC were determined by using a multiple comparison method. No EC_{10, 20, 50}-values were determined due to inhibition below 50 %.

The validity of the test was confirmed.

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 100 mg/L (nominal) and 5.75mg/L (actual). The overall LOEC was therefore determined to be 100 mg/L (nominal) and 5.75 mg/L (actual), the corresponding NOEC was set at 31.3 mg/L (nominal) and 2.06 mg/L (actual).

The EC₂₀- and EC₅₀-value for growth rate and the EC₅₀-value for yield were considered to be > 100 mg/L (nominal) and 5.75 mg/L (actual). Due to an inhibition of growth rate below 20 % and yield below 50 % as well as a missing concentration response relation no reliable values were calculable. The EC₁₀-value for growth rate and the EC_{10, 20}-value for yield were not determined.