Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

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:

16 Jun 2017 - 20 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006, Annex 5 corrected 2011

Deviations:

yes

Remarks:

For the final test, the effect parameters were calculated based on only four concentrations instead of (at least) five as prescribed by the Guideline. This could have effect on the NOEC, and therefore the EC10 is recommended to be used instead of NOEC.

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

purity: 97.3%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, WAFs prepared at 10, 18, 32, 56 and 100 mg/L
- Sampling method: at t=0 h, t=24 h, and 72 h samples of 2.0 mL were taken from all test concnetrations and control. In addition, the filter containing the undissolved residue was kept for possible analysis. Additionally, 2.0 mL reserve samples were taken from all test solutions for possible analysis.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: All test solutions were individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. A one day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were filtered through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10E+04 cells/mL.
- Controls: Test medium without test item or other additives.
- Evidence of undissolved material: Solutions were checked for Tyndall effect with a laser pen. No distraction of the beam was observed besides the highest concentration in the combined limit/range-finding test end the second full test in medium without salts. All test solutions were visually clear and the color ranged from light to dark orange at the end of the preparation procedure.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis)) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C, until the pre-culture started.

PRE-CULTURE
- 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium as used in the Final test: Standard M2 medium, according to OECD 201 Guideline.
- Pre-culture medium as used in the salt-deficient Final tests: Standard M2 medium without calcium and magnesium salts.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Standard M2 medium: 0.24 mmol/L

Test temperature:

22-23 °C

pH:

7.7-8.2

Nominal and measured concentrations:

Nominal: WAFs prepared at at 10, 18, 32, 56 and 100 mg/L.
Initial measured: 0.69, 0.91, 0.93, 2.1 and 18 mg/L. The measured concentrations remained at the level of initially measured during the exposure period (97-110%). See Table 3 in "Any other information on results incl. tables" for measured concentrations over time.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all glass vessels, capped, containing 50 mL of test solution.
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 226 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 or 2 replicates of the highest concentration without algae; 1 or 2 replicates of each test concentration for sampling purposes.
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Medium used: M2 for final test with standard medium; modified M2 (without calcium and magnesium salts) to distinguish between direct toxicity of the test item and indirect effects caused by deprivation of these salts. The test item can react with calcium and magnesium salts.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.
- Intervals of water quality measurement: pH measured at beginning and end of test; temperature of medium continuously measured in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 80 to 85 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: most probably the WAF of 56 mg/L was not inoculated with algae and therefore, one concentration was missing for the determination of effect parameters. Any results for this concentration are removed from further analyses. This could have effect on the NOEC for growth rate and yield inhibition and therefore, the EC10 is recommended to be used instead of NOEC.
- Combined Limit/Range finding study concentrations: Control, WAFs individually prepared at a loading rate of 1.0, 10, 100 mg/L.
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate (test performed Sept 2017).

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% c.i.: 5.7-12

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 18 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes. For the growth curve, see attached illustration.
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest concentration when compared to the control.
- Any stimulation of growth found in any treatment: in tests performed in medium deprived of salts, a stimulation of growth rather than inhibition was observed at the highest concentration when compared to the control treatment. This indicates that the test item compensates for the salts when present in the medium. Therefore, it was concluded that the effects observed in the test performed in the standard medium are due to direct toxicity of the test item.
- Effect concentrations exceeding solubility of substance in test medium: The EC50 for growth rate inhibition (72h-ERC50) was beyond the range tested, i.e. exceeded the highest soluble concentration in test medium, i.e. >18 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- The EC50 for growth rate inhibition (72h-ERC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller. The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 3          
Final Test 1 (Standard Medium): Test Samples

Time of sampling [hours]	Date of sampling	Date of analysis^1	Loading rate^2 [mg/L]	Concentration analyzed [mg/L]	Relative to initial [%]
0	31 Jul 2017	08 Sep 2017	0	n.d.
			10	0.688
			18	0.905
			32	0.932
			56	2.11
			100	17.6
			100^3	17.9
24	01 Aug 2017	08 Sep 2017	0	n.d.	n.a.
			10	0.700	102
			18	0.916	101
			32	0.908	97
			56	2.22	106
			100	19.3	110
			100^3	19.2	107
72	03 Aug 2017	08 Sep 2017	0	n.d.	n.a.
			10	0.687	100
			18	0.902	100
			32	0.930	100
			56	2.26	108
			100	17.1	97
			100^3	16.1	90

¹     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

²     A water accommodated fraction (WAF) prepared at the loading rate.

³     Without algae.

Table4          
Growth Rate And Percentage Inhibition For The Total Test Period in the standard M2 medium

D&C Red 6 Measured conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.805	0.0269	6
0.69	1.822	0.0245	3	-0.9
0.91	1.791	0.0271	3	0.8
0.93	1.815	0.0157	3	-0.5
18	1.488*	0.0198	3	18

* - effect was statistically significant

Validity criteria fulfilled:

yes

Remarks:

For details on the validity criteria, please refer to "Overall remarks" section.

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata D&C Red 6 reduced growth rate and inhibited the yield of this fresh water algae species significantly at a measured concentration of 18 mg/L. The EC50 for growth rate inhibition (72h-ERC50) was beyond the range tested, i.e. exceeded the highest soluble concentration in test medium, i.e. >18 mg/L. The EC10 for growth inhibition (72h-ERC10) was 9.8 mg/L with a 95% confidence interval ranging from 5.7 to 12 mg/L.

Executive summary:

The toxicity of D&C Red 6 to freshwater algae (Pseudokirchneriella subcapitata) was examined in a study according to OECD TG 201 and in compliance with GLP criteria.

Algae, at a starting cell concentration of 1E+04 cell/mL (nominal) were exposed to Water Accommodated Fractions individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The respective measured initial concentrations were determined to be 0.69, 0.91, 0.93, 2.1 and 18 mg/L. These measured concentrations remained at the level of initially measured during the exposure period. The 56 mg/L WAF (2.1 mg/L measured concentration) was removed from analysis since it was probably not inoculated with algae. The concentration of 18 mg/L was considered the highest soluble in medium. Therefore, the effects parameters were expressed in terms of measured concentrations.

After 72 hours exposure, a statistically significant inhibitory effect on the growth rate of algae was seen only in the highest test concentration of 18 mg/L. Based on these findings, the 72-h ErC50 and ErC10 values are determined to be >18 and 9.8 mg/L, respectively (based on initial measured concentrations). The NOErC cannot be accurately calculated due to the missing results for WAF of 56 mg/L. Therefore, the EC10 is recommended to be used instead of NOEC. The validity criteria were met and the study considered to be reliable without restrictions.

Description of key information

The toxicity of D&C Red 6 to freshwater algae (Pseudokirchneriella subcapitata) was examined in a study according to OECD TG 201 and in compliance with GLP criteria.

Algae at a starting cell concentration of 1E+04 cell/mL (nominal) were exposed to Water Accommodated Fractions individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The respective measured initial concentrations were determined to be 0.69, 0.91, 0.93, 2.1 and 18 mg/L. These measured concentrations remained at the level of initially measured during the exposure period. The 56 mg/L WAF (2.1 mg/L measured concentration) was removed from analysis since it was probably not inoculated with algae. The concentration of 18 mg/L was considered the highest soluble in medium. Therefore, the effects parameters were expressed in terms of measured concentrations.

After 72 hours exposure, a statistically significant inhibitory effect on the growth rate of algae was seen only in the highest test concentration of 18 mg/L. Based on these findings, the 72-h ErC50 and ErC10 values are determined to be >18 and 9.8 mg/L, respectively (based on initial measured concentrations). The NOErC cannot be accurately calculated due to the missing results for WAF of 56 mg/L. Therefore, the EC10 is recommended to be used instead of NOEC. The validity criteria were met and the study considered to be reliable without restrictions.