1-(methylamino)anthraquinone

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD No 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-(methylamino)anthraquinone

EC Number:

201-417-2

EC Name:

1-(methylamino)anthraquinone

Cas Number:

82-38-2

Molecular formula:

C15H11NO2

IUPAC Name:

1-(methylamino)anthraquinone

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media (containing algae) of the test concentration 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 concentration during the test period, duplicate samples from the test media of the test concentration and the control (containing algae) at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment. Additional samples were taken at 48 h to track the degradation of the test item during the test.
The samples were diluted by a factor of 1.25 with acetonitrile.
One additional sample of the control and of the dilution solvent was taken at test start, after 48 h and at test end without any sample treatment.

Storage: The samples from test start, and 48 hours were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. The samples from test end were analysed directly after sampling. Afterwards the samples were again stored deep frozen (< -20 °C) and will be kept stored up to the date of the final report.

Test solutions

Vehicle:

no

Details on test solutions:

Dosage of Test Item: The test item was not well soluble in test water. In this study, only the inhibitory effects of dissolved test item was tested and thus no concentrations above the solubility limit of the test item in test water were tested. Therefore, the only test concentration of 10 mg test item/L was prepared by suspending 10.0 mg test item in 1000 mL test water. The solution was stirred for 48 hours at room temperature in the dark to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter).
The test media were prepared just before introduction of the algae (= start of the test).

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
Origin: The algae were supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22 to 23°C

pH:

7.9 to 8.1 at test start and
9.6 to 9.7 at test end

Nominal and measured concentrations:

Test Concentrations:
A filtrate of 10 mg test item/L and a control, corresponding to 3.81 µg/L geometric mean measured concentration of the test item.
Due to the limit of solubility of the test item in the test medium no higher concentrations could be tested.

Details on test conditions:

Water 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 was:
22 to 23 °C
pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH was:
7.9 to 8.1 at test start and
9.6 to 9.7 at test end
Light Regime: Continuous illumination
Light Intensity: 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:
4998 lux (range: 4640 to 5260 lux)
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 3.81 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.381 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 3.81 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static limit test. The highest test concentration was a filtrate of nominal 10 mg test item/L corresponding to a geometric mean measured concentration of 3.81 µg test item/L. Due to the limit of solubility of the test item in the test medium no higher concentrations could be tested. The 72-hour NOEyC and NOErC were determined to be ≥ 3.81 µg test item/L. The 72-hour LOEyC and LOErC were determined to be > 3.81 µg/L and the 72-hour EyC50 and ErC50were determined to be > 3.81 µg/L.
The test item has no toxic effect at its solubility limit.

Reported statistics and error estimates:

The EC50 values were determined directly from the raw data.
For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at the only test concentration were tested for significant differences compared to the control values by Two-sample testing.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Any other information on results incl. tables

Biological Results

Yield y and Percentage Inhibition of y during the Test Period

Geometric Mean Measured Concentration	Yields y [10000 cells/mL] and % inhibition of y
	24 hours			48 hours			72 hours
[µg test item/L]	y	%		y	%		y	%
Control	4.100	-		31.890	-		150.352	-
3.81	4.359	-6.3		34.381	-7.8		153.015	-1.8

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

No statistically significant difference(tested with Two sample t-test,a = 0.05, one-sided)

 

Growth Rates µ and Percentage Inhibition of µ during the Test Period

Geometric Mean Measured Concentration	Growth rates µ [1/day] and % inhibition of µ
	0 - 24 hours			0 - 48 hours			0 - 72 hours
[µg test item/L]	µ	%		µ	%		µ	%
Control	2.219	-		2.085	-		1.903	-
3.81	2.273	-2.4		2.122	-1.8		1.908	-0.3

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

No statistically significant difference(tested with Two sample t-test,a = 0.05, one-sided)

Analytical Results

Sample Description	Geometric mean	RSD
	measured concentration
	[µg test item/L]	[%]	n
Control	n.a.	n.a.	6
Filtrate of 10 mg/L	3.81	92	6

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static limit test. The highest test concentration was a filtrate of nominal 10 mg test item/L corresponding to a geometric mean measured concentration of 3.81 µg test item/L. Due to the limit of solubility of the test item in the test medium no higher concentrations could be tested. The 72-hour NOEyC and NOErC were determined to be ≥ 3.81 µg test item/L. The 72-hour LOEyC and LOErC were determined to be > 3.81 µg/L and the 72-hour EyC50 and ErC50were determined to be > 3.81 µg/L.
The test item has no toxic effect at its solubility limit.
The quantification of the test item in the test samples was performed using liquid chromatography with UV detection.
The concentrations of the test item were determined in the duplicate test media samples from the test concentration (filtrate of 10 mg/L) and the duplicate control samples from all sampling times. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20% of the initial measured concentrations during the test.

Executive summary:

Title:		Test item: Toxicity toPseudokirchneriella subcapitatain an Algal Growth Inhibition Limit Test
Purpose:		The purpose of this test was to determine the inhibitory effect of the test item on the growth of the freshwater green algaePseudokirchneriella subcapitata. Exponentially growing cultures of this unicellular green algal species were exposed to the only test item concentration of a filtrate of nominal 10 mg/L under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. This limit test was performed in compliance with the test guidelines to demonstrate that the test item has no toxic effect onPseudokirchneriella subcapitataup to at least this concentration. The test method of application and the test system are recommended by the test guidelines andPseudokirchneriella subcapitatais one of the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.
Guidelines/Recommendations:		-     OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006, corrected July 28, 2011 -     Commission Regulation (EC) No 761/2009, Annex, Part C, C.3.: "Freshwater Algae and Cyanobacteria, Growth Inhibition Test", Official Journal of the European Union (EN), dated August 24, 2009 -     OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000 -     SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Material and Methods
Test Item:		Solvent Red 111
Test Species:		Pseudokirchneriella subcapitata, Strain No. 61.81 SAG cultivated in the laboratories of ibacon; original source:"Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Test Design:		This study encompassed two treatment groups: one control and a filtrate of a supersaturated stock solution of nominal 10 mg test item/L withsixreplicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated withapprox. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.
Endpoints:		Yield and growth rate of the algae
Test Concentration:		A filtrate of 10 mg test item/L and a control, corresponding to 3.81 µg/L geometric mean measured concentration of the test item. Due to the limit of solubility of the test item in the test medium no higher concentrations could be tested.
Test Conditions:		Water temperature:22to 23 °C; pH values at test start7.9to 8.1, at the end of the test9.6to 9.7; continuous illumination; mean light intensity:4998 lux (4640 to 5260 lux).
Results:

Table 1. Biological Results

Parameter		Yield [µg test item/L]	Growth rate [µg test item/L]
72-hour EC50		>3.81	>3.81
95 % conf. interval		n.d.	n.d.
72-hour NOEC		≥3.81	≥3.81
72-hour LOEC		>3.81	>3.81
n.d. = not determinable Values refer to geometric mean measured test concentrations
Conclusion:	The influence of test item on the growth of the freshwater green algaePseudokirchneriella subcapitatawas assessed in a static limit test. The highest test concentration was a filtrate of nominal 10 mg test item/L corresponding to a geometric mean measured concentration of 3.81 µg test item/L. Due to the limit of solubility of the test item in the test medium no higher concentrations could be tested. The 72-hour NOEyC and NOErC were determined to be ≥ 3.81 µg test item/L. The 72-hour LOEyC and LOErC were determined to be > 3.81 µg/L and the 72-hour EyC50and ErC50were determined to be > 3.81 µg/L. The test item has no toxic effect at its solubility limit. The quantification of the test item Solvaperm-Rot PFS in the test samples was performed using liquid chromatography with UV detection. The concentrations of the test item were determined in the duplicate test media samples from the test concentration (filtrate of 10 mg/L) and the duplicate control samples from all sampling times. All reported results refer to geometric mean concentrations, since the test item concentrations were not within± 20% of the initial measured concentrations during the test.

This study is classified acceptable and satisfies the guideline requirements for growth inhibition algae studies.