Disodium 4,5-dihydroxybenzene-1,3-disulphonate

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:

28 May - 16 Aug 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23. Mar. 2006, Annex 5 corrected: 28 July 2011

Deviations:

yes

Remarks:

See section "Any other information on material and methods incl tables"

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

adopted 07. December 2015

Deviations:

yes

Remarks:

See section "Any other information on material and methods incl tables"

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,5-dihydroxybenzene-1,3-disulphonate

EC Number:

205-741-5

EC Name:

Disodium 4,5-dihydroxybenzene-1,3-disulphonate

Cas Number:

149-45-1

Molecular formula:

C6H6O8S2.2Na

IUPAC Name:

disodium 4,5-dihydroxybenzene-1,3-disulphonate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test item concentrations;
- Sampling method: Sampling at test start and test end

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 500 mL of a stock solution containing 100.0 mg/L (experiment I) and 10.0 mg/L (experiment II) test item in algal medium was prepared. The resulting solution was clear. Lower test concentrations were prepared by dilution of the stock solution in algal medium.
- Controls: Yes, algae medium only

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: 86.81
- Source: Originally obtained in January 2016 from MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen), Germany
- Age of inoculum : 4 day (pre-culture)
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Culturing media and conditions (same as test or not): Same for pre-culture

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Experiment I: 21.6 – 22.6 °C
Experiment II: 19.4 – 23.3 °C (see section "Any other information on material and methods incl tables" for deviation)

pH:

Experiment I:
8.1 - 8.3 (Control)
7.4 - 8.2 (Test item concentrations)
Experiment II:
8.3 - 8.9 (Control)
7.8 - 8.9 (Test item concentrations)

Nominal and measured concentrations:

Nominal: 0.1 / 0.32 / 1 / 3.2 / 10 / 32 / 100 mg/L (experiment I) For details on measured concentrations please refer to table 7 to 10 in section "Any other information on results incl tables".
Nominal: 0.0032 / 0.01 / 0.032 / 0.1 / 0.32 / 1 mg/L (experiment II) For details on measured concentrations please refer to table 7 to 10 in section "Any other information on results incl tables".

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: glass flasks total volume 65 mL, 45 ± 1 mL of the respective test solution
- Initial cells density: 2.1 *10E+03 cells/mL (experiment I and II)
- Control end cells density: 405477 (experiment I); 336973 cells/mL (expermient II)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Others: Shaken on an orbital shaker to keep the algae in suspension

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: algal medium corresponds to the OECD TG 201 medium was producded with deionised sterilized water
- Culture medium different from test medium: stock culture are kept on solid agar; liquid algal medium (corresponds to the OECD TG 201 medium) was used for test and pre-culture

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continous illumination
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: cell number was determined with an electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes, non GLP pre-tests, no information on results given

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval

Remarks:

0.36 – 0.65 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval

Remarks:

0.06 – 0.09 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (geom. mean)

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 algae cells were observed at test item concentrations of 1 - 100 mg/L.

Results with reference substance (positive control):

ErC50 (72 h): 0.79 mg/L, test performed in January 2018

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel ®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1. The details of calculation are stated in chapter 19 Annex 5: Statistical calculation using ToxRat® Professional 3.2.1.

Any other information on results incl. tables

Table 1: Microscopical Observations

Experiment I
Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
0.1	Yes
0.32	Yes
1	No cells visible
3.2	No cells visible
10	No cells visible
32	No cells visible
100	No cells visible
Experiment II
Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
0.0032	Yes
0.01	Yes
0.032	Yes
0.1	Yes
0.32	Yes
1	No cells visible

Treatment 0.0032 mg/L not used for evaluation, as some replicates were turbid

Table 2: Cell Number/mL (experiment I)

Nominal Concentration	Parameter	Cell Number/mL
in mg/L		0 h	24 h	48 h	72 h
Blank control	Mean	2100	11550	74097	405477
Blank control	SD	0	723	3861	29378
0.1	Mean	2100	8187	44100	183800
0.1	SD	0	1110	4568	22795
0.32	Mean	2100	7840	19440	84813
0.32	SD	0	658	658	11680
1	Mean	2100	6673	13133	16167
1	SD	0	873	2487	892
3.2	Mean	2100	7373	13527	13553
3.2	SD	0	670	3606	1497
10	Mean	2100	7680	13140	14487
10	SD	0	500	302	1808
32	Mean	2100	7527	12953	16793
32	SD	0	687	424	884
100	Mean	2100	6080	13893	16320
100	SD	0	677	1230	1311

SD = Standard Deviation

 

Table 3: Cell Number/mL (experiment II)

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2100	12493	57847	336973
Blank control	SD	0	1230	4134	24070
0.0032	Mean	2100	10220	43587	250673
0.0032	SD	0	1849	3507	16372
0.01	Mean	2100	12513	62007	360980
0.01	SD	0	232	3156	8152
0.032	Mean	2100	12207	56987	349200
0.032	SD	0	515	4268	25824
0.1	Mean	2100	10133	41907	251713
0.1	SD	0	1544	4818	26433
0.32	Mean	2100	10033	26393	70013
0.32	SD	0	674	1187	2807
1	Mean	2100	9447	15433	20313
1	SD	0	1467	1305	2066

SD = Standard Deviation; Treatment 0.0032 mg/L not used for evaluation, as some replicates were turbid

  

Table 4: Inhibition Values (experiment I), calculated by Microsoft Excel

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
0.1	15.10	54.96
0.32	29.83	79.49
1	61.22	96.51
3.2	64.64	97.16
10	63.39	96.93
32	60.50	96.36
100	61.07	96.47

 

Table 5: Inhibition Values (experiment II), calculated by ToxRat

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
0.0032	5.81	25.77
0.01	-1.4	-7.2
0.032	-0.7	-3.7
0.1	5.8	25.5
0.32	30.9	79.7
1	55.4	94.6

Negative inhibition values indicate a stimulation of algal growth compared to the blank control; Treatment 0.0032 mg/L not used for evaluation, as some replicates were turbid

 

Table 6: Biological Results of the Test Item

Parameter	Value	95% confidence interval
NOEC (Growth Rate) 72 h	0.02 mg/L	--
NOEC (Yield) 72 h	0.02 mg/L	--
LOEC (Growth Rate) 72 h	0.06 mg/L	--
LOEC (Yield) 72 h	0.06 mg/L	--
72h ErC10	0.07 mg/L	0.06 – 0.09 mg/L
72h EyC10	0.04 mg/L	0.03 – 0.05 mg/L
72h ErC50	0.49 mg/L	0.36 – 0.65 mg/L
72h EyC50	0.11 mg/L	0.07 – 0.15 mg/L

Analytical Determination:

Experiment I

The measured concentrations in treatment 0.1 mg/L were below the LOQ (0.05 mg/L) at the start of the test and after 72 hours. The measured concentrations in treatment 0.32 mg/L were below the LOQ after 72h. Half the LOQ (0.025 mg/L) was used to calculate the geometric mean of the treatment 0.32 mg/L for the 72h value. This is permitted according to Guidance Document No. 23. The measurable concentrations lay between 41 % and 92 % of the nominal concentrations at the beginning of the test and between 50 % and 88 % of the nominal concentrations at the end of the test. The decrease in the recovery of the test item with decreasing concentration can be explained by the adsorption of dissolved test item onto the algae cells.

Experiment II

In treatment 0.0032 mg/L to 0.032 mg/L the measured concentrations at the beginning of the test were below the LOQ. At the end of the test, the concentrations in the treatments 0.0032 - 0.1 mg/L were below the LOQ. The measurable concentrations lay between 58 % and 72 % of the nominal concentrations at the beginning of the test and between 48 % and 58 % of the nominal concentrations at the end of the test and therefore not within ± 20% of the nominal values. Since the lower concentrations were prepared by dilution of the stock solution, the real concentrations were calculated on the basis of the geometric mean of the measured concentration in the highest concentrated treatment and the respective dilution factor. Geometric mean is calculated by multiplication of the n participating concentrations and taking the nth root. At the lowest concentration (0.0032 mg/L), the solution became partly turbid after 48 hours. This treatment is therefore excluded from the evaluation. Turbidity was not caused by the test item because in the 2 next higher concentrations the solutions were not turbid and no inhibition of algal growth was observed.

Table 7: Measured Concentrations and % of Nominal (Experiment I)

Nominal Concentration Test Item	Measured concentration t = 0 h	Measured concentration t = 72 h	% of Nominal t = 0 h	% of Nominal t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	< LOQ	< LOQ	--	--
0.1	< LOQ	< LOQ	--	--
0.32	0.13	< LOQ	41	--
1	0.67	0.50	67	50
3.2	2.57	2.25	80	70
10	8.83	7.78	88	78
32	28.58	25.70	89	80
100	92.39	87.55	92	88

LOQ (Limit of quantification) = 0.05 mg/L

 

Table 8: Measured Concentrations and % of Nominal (Experiment II)

Nominal Concentration Test Item	Measured concentration t = 0 h	Measured concentration t = 72 h	% of Nominal t = 0 h	% of Nominal t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	< LOQ	< LOQ	--	--
0.0032	< LOQ	< LOQ	--	--
0.01	< LOQ	< LOQ	--	--
0.032	< LOQ	< LOQ	--	--
0.1	0.06	<	LOQ	58
0.32	0.21	0.15	65	48
1	0.72	0.58	72	58

LOQ (Limit of quantification) = 0.05 mg/L

Treatment 0.0032 mg/L not used for evaluation, as some replicates were turbid

 

Table 9: Geometric Mean (Experiment I)

Nominal Concentration Test Item	Geometric Mean
mg/L	mg/L
Blank control	--
0.1	--
0.32	0.06
1	0.58
3.2	2.40
10	8.29
32	27.10
100	89.93

  

Table 10: Geometric Mean and calculated concentrations (Experiment II)

Nominal Concentration Test Item	Geometric Mean	Calculated concentrations
mg/L	mg/L	mg/L
Blank control	--	--
0.0032	--	0.002
0.01	--	0.006
0.032	--	0.021
0.1	0.058	0.065
0.32	0.179	0.207
1	0.648	0.648

Treatment 0.0032 mg/L not used for evaluation, as some replicates were turbid

Table 10: 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.	193 (experiment I) 160 (experiment II)	Yes 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%	6%(experiment I) 9%(experiment II)	Yes 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%(experiment I) 1%(experiment II)	Yes Yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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