Carboxylic acids, di-, C5-9

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:

25 - 28 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996

GLP compliance:

yes (incl. QA statement)

Remarks:

National Institute of Pharmacy and Nutrition, Budapest, Hungary

Test material

Test material information

Constituent 1

Reference substance name:

Carboxylic acids, di-, C5-9

EC Number:

701-329-9

Molecular formula:

C5di: C5H8O4 C6di: C6H10O4 C7di: C7H12O4 C8di: C8H14O4 C9di: C9H16O4

IUPAC Name:

Carboxylic acids, di-, C5-9

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: applied test concentration levels and from the control at the beginning and at the end of the experiment
- Sampling method: 2 mL sampled in glass tubes
- Sample storage conditions before analysis: samples were frozen and kept approximately at -20°C

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: saturated solution method (water accommodated fraction, WAF), test solutions were prepared individually by dispersing/dissolving the amount of test item into the test medium two days before the start of the experiment, shaken for about 24 hours at approximately 30°C, then equilibrated for about 24 hours at approximately 20°C, non-dissolved test material was removed by filtration (0.22 µm) to give the appropriate WAF solutions.
- Differential loading: yes
- Controls: Algal growth medium inoculated with algal cells
- Evidence of undissolved material: test item was stable in media

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: 61.81 SAG
- Source: supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology,and Universityof Göttingen, Germany
- Age of inoculum: pre-culture incubated about three days
- Method of cultivation: Stock cultures are small algal colonies inoculated onto agar regularly, transferred to fresh agar medium at least once every two months and maintained under standardised conditions according to the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.4 - 22.6°C

pH:

5.15 - 8.41

Nominal and measured concentrations:

Nominal loading rate: 0 (control), 6.25, 12.5., 25.0, 50.0 and 100.0 mg/L
Mean measured concentrations: 0 (control), 6.28, 12.36, 24.52, 48.12 and 93.36 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: covered with air-permeable stoppers
- Fill volume: 100 mL
- Initial cells density: 10^4 cells/mL
- Control end cells density: 67.33 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (Reconstituted algal growth medium (OECD medium, according to OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature (beginning of the experiment and each day thereafter in a flask filled with water in climate chamber), pH (beginning and at the end of the test, in the control and each concentration)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 7922 lux (equivalent to ~107 µE/m2/s), fluorescent lamps (400-700 nm)

EFFECT PARAMETERS MEASURED: cell numbers and abnormal appearance (24, 48 and 72 hours after test start),
- Determination of cell concentrations: microscopic method with a counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: control, 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Average of cell number at 72 hours (x 104cell/mL): 71.33 (control), 70.50 (0.1 mg/L), 71.00 (1 mg/L), 70.00 (10 mg/L) and 1.00

Reference substance (positive control):

yes

Remarks:

Potassium dichromate tested at least twice a year

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

47.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95%-confidence limits: 43.42 – 52.19 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

23.63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95%-confidence limits: 20.66 – 27.02

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

24.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Thin cells were observed at the two highest examined concentrations of 50.0 and 100.0 mg/L nominal loading rates (WAFs)

Results with reference substance (positive control):

- Results with reference substance valid? yes
72h ErC 50: 0.82 mg/L, (95 % confidence limits: 0.76 – 0.90 mg/L)

Reported statistics and error estimates:

ErL/ErC, EbL/EbC and EyL/EyC values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. Statistical comparisons of biomass, average specific growth rates and yield in controls
and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. For the determination of the LOELR/LOEC and NOELR/NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were compared with controls using the Bonferroni t-Test (α = 0.05) by TOXSTAT software.

Any other information on results incl. tables

Table 1: Measured exposure concentrations

Concentration (mg/L nominal loading rate WAF)	Measured concentrations (mg/L)		Geometric mean
	0h/start	72h/end	mg/L
6.25	6.34	6.22	6.28
12.5	12.13	12.60	12.36
25.0	24.37	24.67	24.52
50.0	47.67	48.57	48.12
100.0	93.23	93.50	93.36

Table 2: Cell Number (x 10^4 cells/mL)

 

Concentration [mg/L nominal loading rate WAF]		Number of cells
	0 h	24 h	48 h	72 h
	1	4	17	75
	1	4	18	65
	1	4	18	67
Control	1	3	17	66
	1	5	20	68
	1	5	17	63
Mean	1.00	4.17	17.83	67.33
SD	0.0	0.8	1.2	4.1
	1	3	17	66
	1	5	17	65
6.25	1	3	17	62
Mean	1.00	3.67	17.00	64.33
SD	0.0	1.2	0.0	2.1
	1	3	17	60
	1	4	18	64
12.5	1	3	16	62
Mean	1.00	3.33	17.00	62.00
SD	0.0	0.6	1.0	2.0
	1	3	10	48
	1	3	11	50
25.0	1	4	12	51
Mean	1.00	3.33	11.00	49.67
SD	0.0	0.6	1.0	1.5
	1	4	4	9*
	1	3	3	11*
50.0	1	3	3	12*
Mean	1.00	3.33	3.33	10.67
SD	0.0	0.6	0.6	1.5
	1	1	2*	2*
	1	1	1*	1*
100.0	1	1	1*	1*
Mean	1.00	1.00	1.33	1.33
SD	0.0	0.0	0.6	0.6

*  : Thin, deformed cells

Table 3: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	67.33	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	11.59%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1.42%	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.