Reaction mass of 3,4,5-trichlorophthalic acid, strontium salt and 3,4,6-trichlorophthalic acid, strontium salt and 3,4-dichlorophthalic acid, strontium salt and 3,5-dichlorophthalic acid, strontium salt and 3,6-dichlorophthalic acid, strontium salt and 3-chlorophthalic acid, strontium salt and 4,5-dichlorophthalic acid, strontium salt and 4-chlorophthalic acid, strontium salt

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:

2019-09-17 to 2019-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted March 2006, corrected July 28, 2011, equivalent to Commission Regulation (EC) NO 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Filtrate of 100 mg test item/L representing the highest test concentration and dilutions of 1:3.125, 1:10, 1:31.25 and 1:100 of this filtrate was tested
- Sampling method: The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
- Sample storage conditions before analysis: All samples from test start were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed, samples from test end were analyzed directly after sampling. Afterwards all samples were stored deep frozen (≤ -20 °C) and will be kept stored up to the date of the final report.

Vehicle:

no

Details on test solutions:

The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 100.6 mg test item into 1006 mL test water by intense stirring for 25 minutes and ultrasonic treatment for 5 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the algae (= start of the test).
-Eluate: N/A
-Differential loading: N/A
-Controls: In the control, test water was used without addition of the test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle used
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s): N/A
- Evidence of undissolved material (e.g. precipitation, surface film, ect.): There were no remarkable observations in the test medium.

Test organisms (species):

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

Details on test organisms:

- Common name: Pseudokirchneriella subcapitata (KORSHIKOV) formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen (SAG)", 37073 Göttingen, Germany.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardized conditions according to the test guidelines.
Reference Item: For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): same as test.
- Any deformed or abnormal cells observed: No.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature: 21.4 to 22.0 °C

pH:

The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH value in the control at test start: 8.1 to 9.1 at test end.

Nominal and measured concentrations:

At the start of the test 95 % of the nominal test concentrations were found (average of the nominal concentrations of 100, 32, 10 and 3.2 mg test item/L). After 72 hours test duration, 96 % of the nominal value was determined (average of the nominal concentrations of 100, 32, 10 and 3.2 mg test item/L). During the test the algae were exposed to a mean of 95 % of nominal.

Details on test conditions:

TEST SYSTEM
-Test vessel: Erlenmeyer flasks
-Type: open
-Material, size, headspace, fill volume: 50 mL volume with nominal 50 mL of test medium
-Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): No, static
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 5000 algal cells per mL test medium; These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.
- Control end cells density: 814 770 000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):6
- No. of vessels per vehicle control (replicates): N/A
The test was performed with three replicates per test concentration and six replicates in the control. Volumes of 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass stoppers and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.
Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item.
GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water, Analytical grade salts were added to achieve OECD medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: Start and end; Test conditions were recorded with suitable instruments and documented.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5034 lux (range: 4815 to 5190 lux)
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.
- Chlorophyll measurement: No

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Justification for using less concentrations than requested by guideline: N/A

Range finding study
- Test concentrations: test medium of 100 mg/L and dilutions thereof of 1:3.126, 1:10 1:31.25 and 1:100 and a control
- Results used to determine the conditions for the definitive study: Yes
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions. The pre-experiments were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study number of the present study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

not determinable because of methodological limitations

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

not determinable because of methodological limitations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

not determinable because of methodological limitations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

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. The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.
- Unusual cell shape: No
- Color differences: No
- Flocculation: No
- Adherence to test vessels: No
- 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: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 0.941 mg test item/L (95 % C.I.: 0.910 - 0.973 mg test item/L)

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams Multiple Sequential t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Biological results:

The 72-hour EC₅₀ for yield and growth rate was calculated to be >100 mg test item/L. The 72-hour E_yC₁₀was calculated to be > 100 mg test item/L and the E_rC₁₀> 100 mg test item/L. The 72-hour NOE_yC was determined to be 32 mg test item/L and the associated 72-hour LOE_yC of 100 mg test item/L. The 72-hour NOE_rC was determined to be 32 mg test item/L and the associated 72-hour LOE_rC is 100 mg test item/L.

Table 1: Influence of Strontium Dichlorophthalate on the Growth of Pseudokirchneriella subcapitata. The reported results refer to nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test.

Parameter	Yield [mg test item/L]	Growth rate [mg test item/L]
72-hour EC50	>100	>100
95 % conf. interval	n.d.	n.d.
72-hour EC20	>100	>100
95 % conf. interval	n.d.	n.d.
72-hour EC10	> 100	>100
95 % conf. interval	n.d.	n.d.
72-hour NOEC	32	32
72-hour LOEC	100	100
n.d. = not determinable Values refer to nominal test concentrations

Analytical Results:

The quantification of the test item strontium dichlorophthalate in the test samples was performed using liquid chromatography with UV detection. At the start of the test 95 % of the nominal test concentrations were found (average of the nominal concentrations of 100, 32, 10 and 3.2 mg test item/L). After 72 hours test duration, 96 % of the nominal value was determined (average of the nominal concentrations of 100, 32, 10 and 3.2 mg test item/L). During the test the algae were exposed to a mean of 95 % of nominal.

Validity criteria of the test:

Cell Density Increase in Control Cultures:	163.0-fold increase within 72 hours and thus, the validity criterion was met.
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures:	12.7 % and thus, the validity criterion was met.
Coefficient of Variation of Average Growth between Control Replicates:	0.3 % and thus, the validity criterion was met.

Table 2: Algal Cell Densities during the Test Period of 72 Hours

Nominal concentration [mg test item/L]	Flask no.	Density of algal cells [10000/mL] after
		24 hours	48 hours	72 hours
Control	1	2.709	18.192	81.795
	2	2.709	19.145	79.889
	3	3.185	19.860	83.224
	4	2.947	18.192	81.795
	5	2.947	19.384	79.651
	6	3.185	18.192	82.510
	m	2.947	18.828	81.477
	s	0.213	0.733	1.427
1.0	1	2.709	20.098	84.892
	2	3.185	20.813	83.701
	3	3.185	18.192	86.083
	m	3.026	19.701	84.892
	s	0.275	1.355	1.191
3.2	1	2.470	16.763	79.651
	2	2.709	18.907	83.939
	3	2.709	18.431	80.604
	m	2.629	18.034	81.398
	s	0.138	1.126	2.251
10	1	3.185	20.336	89.656
	2	2.947	20.575	85.606
	3	2.947	19.384	77.031
	m	3.026	20.098	84.098
	s	0.138	0.630	6.446
32	1	3.185	19.860	82.510
	2	2.709	19.145	81.080
	3	2.709	18.669	74.887
	m	2.867	19.225	79.492
	s	0.275	0.599	4.052
100	1	2.709	16.763	70.123
	2	2.470	14.143	75.840
	3	2.470	15.810	69.884
	m	2.550	15.572	71.949
	s	0.138	1.326	3.372
m: mean value; s: standard deviation At test start 5000 algal cells/mL were inoculated

 

Table 3: Yield during the test period of 72 hours

Nominal concentration [mg test item/L]	Flask no.	Yield Y [10000 cells/mL] after
		24 hours	48 hours	72 hours
Control	1	2.209	17.692	81.295
	2	2.209	18.645	79.389
	3	2.685	19.360	82.724
	4	2.447	17.692	81.295
	5	2.447	18.884	79.151
	6	2.685	17.692	82.010
	m	2.447	18.328	80.977
	s	0.213	0.733	1.427
1.0	1	2.209	19.598	84.392
	2	2.685	20.313	83.201
	3	2.685	17.692	85.583
	m	2.526	19.201	84.392
	s	0.275	1.355	1.191
3.2	1	1.970	16.263	79.151
	2	2.209	18.407	83.439
	3	2.209	17.931	80.104
	m	2.129	17.534	80.898
	s	0.138	1.126	2.251
10	1	2.685	19.836	89.156
	2	2.447	20.075	85.106
	3	2.447	18.884	76.531
	m	2.526	19.598	83.598
	s	0.138	0.630	6.446
32	1	2.685	19.360	82.010
	2	2.209	18.645	80.580
	3	2.209	18.169	74.387
	m	2.367	18.725	78.992
	s	0.275	0.599	4.052
100	1	2.209	16.263	69.623
	2	1.970	13.643	75.340
	3	1.970	15.310	69.384
	m	2.050	15.072	71.449
m: mean value; s: standard deviation

 

Table 4: Growth rate during the Test Period of 72 Hours

Nominal concentration [mg test item/L]	Flask no.	Growth rates [µ/day] after
		0 - 24 hours	0 - 48 hours	0 - 72 hours
Control	1	1.690	1.797	1.699
	2	1.690	1.823	1.691
	3	1.852	1.841	1.705
	4	1.774	1.797	1.699
	5	1.774	1.829	1.690
	6	1.852	1.797	1.702
	m	1.772	1.814	1.698
	s	0.072	0.019	0.006
1.0	1	1.690	1.847	1.712
	2	1.852	1.864	1.707
	3	1.852	1.797	1.716
	m	1.798	1.836	1.711
	s	0.094	0.035	0.005
3.2	1	1.598	1.756	1.690
	2	1.690	1.816	1.708
	3	1.690	1.804	1.694
	m	1.659	1.792	1.697
	s	0.053	0.032	0.009
10	1	1.852	1.853	1.730
	2	1.774	1.859	1.714
	3	1.774	1.829	1.679
	m	1.800	1.847	1.708
	s	0.045	0.016	0.026
32	1	1.852	1.841	1.702
	2	1.690	1.823	1.696
	3	1.690	1.810	1.670
	m	1.744	1.825	1.689
	s	0.094	0.016	0.017
100	1	1.690	1.756	1.648
	2	1.598	1.671	1.674
	3	1.598	1.727	1.647
	m	1.628	1.718	1.656
	s	0.053	0.043	0.015
m: mean value; s: standard deviation

 

 

Table 5: Yield y and Percentage Inhibition of y during the Test Period

Nominal concentration [mg test item/L]	Yields y [10000 cells/mL] and % inhibition of y during the test period
	0 - 24 hours		0-48 hours		0-72 hours
	y	%	y	%	y	%
Control	2.447	-	18.328	-	80.977	-
1.0	2.526	-3.2	19.201	-4.8	84.392	-4.2
3.2	2.129	13.0	17.534	4.3	80.898	0.1
10	2.526	-3.2	19.598	-6.9	83.598	-3.2
32	2.367	3.2	18.725	-2.2	78.992	2.5
100	2.050	16.2*	15.072	17.8*	71.449	11.8*
negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control * mean value significantly different from the control (tested with Williams Multiple Sequential t-test (24, 48 and 72h),α= 0.05, one-sided smaller)

 

Table 6: Growth Rates µ and Percentage Inhibition of µ during the Test Period

Nominal concentration [mg test item/L]	Growth rates µ [1/day] and % inhibition of µ during the test period
	0 - 24 hours		0-48 hours		0-72 hours
	µ	%	µ	%	µ	%
Control	1.772	-	1.814	-	1.698	-
1.0	1.798	-1.5	1.836	-1.2	1.711	-0.8
3.2	1.659	6.4	1.792	1.2	1.697	0.0
10	1.800	-1.6	1.847	-1.8	1.708	-0.6
32	1.744	1.6	1.825	-0.6	1.689	0.5
100	1.628	8.1*	1.718	5.3*	1.656	2.5*

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control

* mean value significantly different from the control (tested with Williams Multiple Sequential t-test (24, 48 and 72h), α = 0.05, one-sided smaller) 

 

Table 7: Summary of Analytical Results

Sample description [mg test item/mL]	% of nominal^1	RSD [%]	n
Control	n.a.	n.a.	4
3.2	92	2	4
10	96	2	4
32	97	1	4
100	98	1	4

¹mean value of all measured samples per treatment group

RSD: relative standard deviation per treatment group

n: number of analysed samples; n.a.: not applicable

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EC50 for yield and growth rate were calculated to be > 100 mg test item/L. The 72-hour NOEC for yield and growth rate was determined to be 32 mg test item/L and the associated 72-hour LOEC for yield and growth rate was 100 mg test item/L. Growth inhibition at 100 mg/L was 2.5 % compared to the control group.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata (KORSHOKOV), Strain No. 61.81 were exposed to Strontium dichlorophthalate at nominal concentrations of 100, 32, 10, 3.2 and 1.0 mg test item/L under static or renewal conditions in accordance with the OECD guideline 201.  The NOEC and EC₅₀ values based on Yield and growth rate were 32 mg test item/L and <100 mg test item, respectively.  The % yield inhibition in the treated algal culture as compared to the control ranged from -4.2 % in the lowest treatment concentration to 11.8% in the highest tested concentration after 72 hours, and the % growth inhibition in the treated groups compared to the control group ranged from -0.8 to 2.5 %, respectively.

This toxicity study is classified as acceptable and satisfies the guideline requirements for Freshwater alga and Cyanobacteria, Growth Inhibition test with P. subcapitata.

Results Synopsis

 

Test Organism: of Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG

Test Type (Flowthrough, Static, Static Renewal): Static

 

Parameter	Yield [mg test item/L]	Growth rate [mg test item/L]
72-hour EC50	>100	>100
95 % conf. interval	n.d.	n.d.
72-hour EC20	>100	>100
95 % conf. interval	n.d.	n.d.
72-hour EC10	>100	>100
95 % conf. interval	n.d.	n.d.
72-hour NOEC	32	32
72-hour LOEC	100	100
n.d. = not determinable

 

Endpoint(s) Effected: Growth rate, Yield

Description of key information

Strontium dichlorophthalate was tested on the freshwater algae Pseudokirchneriella subcapitata according to OECD guideline No. 201. The tested concentrations were 100, 32, 10, 3.2 and 1.0 mg test item/L, and a control. The result of the study was a EC50 of > 100 mg test item/L and a NOEC of 32 mg test item/L based on the growth rate.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

32 mg/L