2-Butenedioic acid (Z)-, ester with 1,2-propanediol, compd. with 2-(dibutylamino)ethanol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to international guideline(s), GLP-compliant, performed in recognized contract research organization, no restrictions, fully adequate for assessment.

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 100 , 50, 25, 12.5, 6.25 mg/L and control
- Sampling intervals: start and end of the test (0 and 72 h)
- Sampling method: samples were taken from flasks containing no algal cells
- Sample storage conditions before analysis: no storage prior to analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The highest test concentration of 100 mg/L and dilutions thereof were prepared in test medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, GERMANY.
- Age of inoculum (at test initiation): Three days. Algal cells used for inoculation were in the exponential phase of growth.
- Method of cultivation: Algae are cultured under standardised conditions. The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 10^7 cells/mL.

ACCLIMATION
- Acclimation period: 3 days (pre-culturing)
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3 (calulated)

Test temperature:

22.4-23.1 °C (daily measurements in a flask filled with water and incubated under the test conditions)
21.8-23.4 °C (continuous measurements in the climatic chamber)

pH:

test start (0 h): 7.7-7.9
test end (72 h): 8.0-9.3
The pH did not increase by more than 1.5 units in the control and in the treatments

Nominal and measured concentrations:

nominal concentrations: 100, 50, 25, 12.5, and 6.25 mg/L
measured concentrations:
start:82-97 % of nominal
end (72 h): 109-134% of nominal (115-147% of initially measured)
The analytically measured concentrations deviated more than 20% from the nominal values in some cases, thus the biological results are based on the geometric mean measured concentrations: 101.9, 51.9, 24.1, 12.2, and. 6.9 mg/L (96-110% of nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks containing 100 mL of inoculated test medium and covered with air-permeable stoppers
- Aeration/gaseous exchange: continuous shaking on an laboratory orbital shaker
- Initial cells density: 1x10⁴ cells/mL
- Control end cells density: 70 x10⁴ cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Ca/Mg ratio: 1:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: about 8211 lux (equivalent to 110 µE/m2/s), provided by fluorescent lamps (spectral range 400-700 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell numbers counted at 24, 48 and 72 hours
- Determination of cell concentrations: counting chamber
- Algal cells were observed microscopically for abnormal appearance at 24, 48 and 72 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study
- Test concentrations: nominal 100, 10, 1 and 0.1 mg/L
- Results used to determine the conditions for the definitive study: inhibition of algal growth (yield) after 72 hours was 85% at 100 mg/L; no significant inhibition was observed at the lower concentrations (≤ 5%)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

91 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 72-115 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

(yield)

Remarks on result:

other: 95% confidence limits: 26-33 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: 32-40 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

24.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: no

Control cultures:
- increase of biomass by a factor of: 70
- mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3): 12%
- coefficient of variation for the average specific growth rates during the test period (day 0-3): 0.85%

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-hour ErC50: 0.94 mg/L (95% confidence limits. 0.85-1.03 mg/L)

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) to demonstrate exponential growth for the entire study period.

The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

The ErC50, EbC50 and EyC50 values of the test item were determined using Probit analysis by TOXSTAT software.

Statistical comparisons of biomass, average specific growth rates and yield in control and in 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 LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Table 1: Cell Number

 

Measured geometric mean and nominal concentration (mg/L)	Replicate number	Cell number (x 10^4/mL)
		24 hours	48 hours	72 hours
Control	R1	4	20	74
	R2	4	17	68
	R3	3	17	67
	R4	5	21	69
	R5	4	20	71
	R6	4	19	71
	Mean	4.0	19.0	70.0
	SD	0.6	1.7	2.5
6.9 (6.25)	R1	4	18	68
	R2	5	19	70
	R3	4	18	67
	Mean	4.3	18.3	68.3
	SD	0.6	0.6	1.5
12.2 (12.5)	R1	5	18	68
	R2	3	16	66
	R3	4	20	70
	Mean	4.0	18.0	68.0
	SD	1.0	2.0	2.0
24.1 (25)	R1	5	18	34
	R2	3	16	28
	R3	3	16	27
	Mean	3.7	16.7	29.7
	SD	1.2	1.2	3.8
51.9 (50)	R1	3	7	12
	R2	3	7	16
	R3	3	8	15
	Mean	3.0	7.3	14.3
	SD	0.0	0.6	2.1
101.9 (100)	R1	2	4	8
	R2	3	6	14
	R3	2	4	8
	Mean	2.3	4.7	10.0
	SD	0.6	1.2	3.5

 

R1-R6: replicate number

Note: the initial cell density was estimated to be 1 x 10⁴/mL

 

 

Table 2  Inhibition of growth

 

Parameter	Measured geometric mean and nominal concentration  (mg/L)	Sample size	Mean	Inhibition (%)
Area under curve 0 to 72 hours	Control	6	1333	N/A
	6.9 (6.25)	3	1304	2.1
	12.2 (12.5)	3	1284	3.6
	24.1 (25)	3	784*	41.1
	51.9 (50)	3	360*	73.0
	101.9 (100)	3	228*	82.9
Growth rate 0 to 24 hours	Control	6	0.0573	N/A
	6.9 (6.25)	3	0.0609	-6.2
	12.2 (12.5)	3	0.0569	0.8
	24.1 (25)	3	0.0529	7.8
	51.2 (50)	3	0.0458	20.1
	101.9 (100)	3	0.0345*	39.8
Growth rate 0 to 48 hours	Control	6	0.0613	N/A
	6.9 (6.25)	3	0.0606	1.1
	12.2 (12.5)	3	0.0601	1.9
	24.1 (25)	3	0.0586	4.4
	51.9 (50)	3	0.0415*	32.3
	101.9 (100)	3	0.0317*	48.3
Growth rate 0 to 72 hours	Control	6	0.0590	0.0
	6.9 (6.25)	3	0.0587	0.6
	12.2 (12.5)	3	0.0586	0.7
	24.1 (25)	3	0.0470*	20.3
	51.9 (50)	3	0.0369*	37.5
	101.9 (100)	3	0.0315*	46.7
Yield	Control	6	69.0	N/A
	6.9 (6.25)	3	67.3	2.4
	12.2 (12.5)	3	67.0	2.9
	24.1 (25)	3	28.7*	58.5
	51.9 (50)	3	13.3*	80.7
	101.9 (100)	3	9.0*	87.0

 

* : statistically significantly different compared to the control values (Bonferroni t-Test;α= 0.05).

 

Validity criteria fulfilled:

yes

Remarks:

Increase of biomass >16 after 72 hours; mean coefficient of variation for section-by-section specific growth rate <35%; coefficient of variation for average specific growth rate <7%.

Conclusions:

The test material is toxic to freshwater algae with a 72-hour ErC50 between 10 and 100 mg/L (calculated value: 91 mg/L).

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h (based on geometric mean concentrations) (OECD 201, EU C.3):

EC50: 91 mg/L, NOEC: 12.2 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

91 mg/L

EC10 or NOEC for freshwater algae:

12 mg/L

Additional information

The results indicate that the test material is toxic to freshwater algae with a 72-hour ErC50 between 10 and 100 mg/L (calculated value: 91 mg/L).