2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine

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:

05 June 2019 to 08 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine

EC Number:

831-167-5

Cas Number:

2126827-44-7

Molecular formula:

C92H106N8O8

IUPAC Name:

2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 2715/18/01
- Expiration date of the lot/batch: 01 July 2020
- Purity: 99%
- CAS number: 2126827-44-7
- Water solubility: determined to be <1µg/L (i.e. < LOD), using the column elution method (Test Facility Reference No. 20182217).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from the light
- Stability under storage conditions: stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: not determined

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples were taken from the vessels at time 0, 24, and 72 hours. 1ml was taken from the approximate centre of the test vessels.
- Sample storage conditions before analysis: The samples taken from the test solutions were analyzed on the day of sampling.
- Stock sampling: In addition, 1.0 mL triplicate samples were taken from the stock solutions in THF (incl. blank THF for treatment of the solvent control) used in the preparation of test solutions for verification of the nominally dosed test item concentration.

Test solutions

Vehicle:

yes

Remarks:

THF

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a stock solution of test item in tetrahydrofuran (THF) at test substance concentration of nominal concentration 0.10 mg/mL. No other treatment than vigorous shaking was needed to completely dissolve the test item. Subsequently, 25 µL stock solution was spiked to 500 mL test medium in order to obtain a final exposure concentration of nominally 5.0 µg/L .
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): tetrahydrofuran (THF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.05ml/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Test solutions were clear and colorless at the end of the preparation procedure. No undissolved test item was observed in the stock or test solution.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: single celled algae
- Source (laboratory, culture collection): In house laboratory culture
- Age of inoculum (at test initiation) and method of cultivation: a stock culture was prepared by inoculating growth medium 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. Three 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 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test.
- Any deformed or abnormal cells observed: not stated

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

not stated

Test temperature:

23°C

pH:

7.8 to 8.3

Dissolved oxygen:

not stated

Salinity:

not measured

Conductivity:

not measured

Nominal and measured concentrations:

Nominal: 5µg/l
Measured: No test item could be quantified in any of the samples taken from the test solutions throughout the test due to matrix effects. According to expert judgement, the used techniques were state-of-the-art and improvement of the analytical method is considered not possible. The measured concentrations of triplicate samples taken from the 0.10 mg/mL stock solution were 0.10, 0.093, and 0.087 mg/mL (each being a mean of 5 runs). The mean concentration was calculated to be 0.094 mg/L, indicating proper preparation of the stock and thus also test solutions. Therefore, effect parameters were expressed in terms of nominal concentrations. No test item was detected in blank THF used in the treatment of the solvent control.

Details on test conditions:

TEST SYSTEM
- Test vessel:100ml all glass vessels with aluminium caps, perforated for ventilation, containing 50ml of test solution.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50ml of test medium with test item per vessel, 50ml headspace
- Aeration: not stated, continuous shaking
- Initial cells density: 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL
- Control end cells density: 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL
- No. of vessels per concentration (replicates): 6 replicates
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per vehicle control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: Yes, M2 medium.
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Ca/mg ratio:not measured
- Conductivity: not measured
- Culture medium different from test medium: stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. This culture was stated in M1 medium according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis. The pretest culture was cultured 3 days before the test, using cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. M2 medium was used.

OTHER TEST CONDITIONS
- Sterile test conditions: not stated
- Adjustment of pH:no
- Photoperiod:continuous illumination
- Light intensity and quality:74-79 µE.m-2.s-1 (second limit test).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the start of the test the 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 immersion probe (path length = 10 mm). At the end of the final test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae compared to the blank control.
- Chlorophyll measurement:not measured

TEST CONCENTRATIONS
- Initial preliminary study: A first limit test was performed of which the results complied with all validity criteria. However, actual exposure concentrations could not be confirmed. Therefore, a second limit test was performed during which new samples were taken and analysed. Test procedure and conditions used in the first limit test were similar to those applied in the second limit test (as detailed in this study record).
- Test concentrations: 5µg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: confirmation of stock concentration was used to confirm nominal exposure conc.

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: confirmation of stock concentration was used to confirm nominal exposure conc.

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Same result for EC10, EC20 and EC50

Remarks:

confirmation of stock concentration was used to confirm nominal exposure conc.

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: Same result for EC10, EC20 and EC50

Remarks:

confirmation of stock concentration was used to confirm nominal exposure conc.

Details on results:

- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the blank control.
- Effect concentrations exceeding solubility of substance in test medium: The limit test concentration was run at a concentration at least 5 times in exceedance of the limit of solubility.

Results with reference substance (positive control):

- EC50: The 72h-EC50 for growth rate inhibition (ERC50) was 2.02 mg/L with a 95% confidence interval ranging from 2.00 to 2.04 mg/L.
- Other: The 72h-ERC50 was within the historical range of 0.82 and 2.60 mg/L, which is based on reference tests performed at the Test Facility during the last ten years. The 72h-ERC50 was not within the range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012. Nevertheless, the test was considered acceptable as the comparison to the most recent historical data was considered more indicative for the sensitivity of the test system.

Reported statistics and error estimates:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.
For determination of the NOEC the approach recommended in the OECD Test Guideline 201 was used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test, α=0.05, one-sided, smaller).
The ECx-values could not be determined because limit tests were performed and the test item proved to be non-toxic (EC50 > maximum concentration tested).

Any other information on results incl. tables

Table 1       
Growth Rate and Percentage Inhibition for the Total Test Period - Second Limit Test

Test item* Nominal concentration (µg/L)	Mean	Std. Dev.	n	%Inhibition
Pooled control	1.850	0.0311	12
5.0	1.842	0.0232	6	0.45

* 2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine.

Table 2       
Growth Rate and Percentage Inhibition at Different Time Intervals - Second Limit Test

 

Test item* Nominal conc. (µg/L)	n	0 – 24 h		24 – 48 h		48 – 72 h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Pooled control	12	2.272		1.820		1.458
5.0	6	2.265	0.32	1.851	-1.7	1.409	3.4

* 2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine.

Table 3:     
Yield and Percentage Inhibition for the Total Test Period - Second Limit Test

Test item* Nominal concentration (µg/L)	Mean	Std. Dev.	n	%Inhibition
Pooled control	257.4	24.02	12
5.0	250.5	17.84	6	2.7

* 2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine.

Table 4 -

Second Limit Test: Stock Solution analysis results

Time of sampling [hours]	Date of sampling	Date of analysis^1	Concentration [mg/mL]			Relative to nominal [%]	Mean relative to nominal (CV) [%]
			Nominal	Analyzed	Mean Analyzed
0	04 Nov 2019	05 Nov 2019	0-THF	<4x10^-6	<4x10^-6 (2)	n.a.	n.a.
			0-THF	<4x10^-6		n.a.	(n.a.)
			0-THF	<4x10^-6		n.a.
			0-THF	<4x10^-6		n.a.
			0-THF	<4x10^-6		n.a.
			0.10	0.109	0.102	109	102
			0.10	0.105		105	(10)
			0.10	0.0933		93
			0.10	0.0889		89
			0.10	0.113		113
			0-THF	<4x10^-6	<4x10^-6 (2)	n.a.	n.a.
			0-THF	<4x10^-6		n.a.	(n.a.)
			0-THF	<4x10^-6		n.a.
			0-THF	<4x10^-6		n.a.
			0-THF	<4x10^-6		n.a.
			0.10	0.0935	0.0925	94	92
			0.10	0.0848		85	(8.0)
			0.10	0.0892		89
			0.10	0.105		105
			0.10	0.0904		90
			0-THF	<4x10^-6	<4x10^-6 (2)	n.a.	n.a.
			0-THF	<4x10^-6		n.a.	(n.a.)
			0-THF	<4x10^-6		n.a.
			0-THF	<4x10^-6		n.a.
			0-THF	<4x10^-6		n.a.
			0.10	0.0780	0.0871	78	87
			0.10	0.0887		89	(11)
			0.10	0.102		102
			0.10	0.0821		82
			0.10	0.0843		84

¹     Samples were treated and analyzed on 04 Nov 2019. Due to system problems, samples were reanalyzed on 05, 07 or 08 Nov 2019. Samples were stored at room temperature.

²     Any response measured in the samples was below the concentration of the lowest calibration solution of the bracket (i.e. 1.0 µg/L). The concentration was reported as <4x10^-6mg/mL taking the dilution factor of 4 into account.

n.a.Not applicable.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of 2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine to Raphidocelis subcapitata over 72 hours resulted in an EC50 of >5µg/Lfor both growth rate and yield.

Executive summary:

A study was carried out to evaluate the toxicity of 2,9,16,23 -tetrakis(2,5 -di-tert-butyl-4 -methoxyphenoxy)phthalocyanine to the algae Raphidocelis subcapitata. The study was based on the OECD 201 Guidelines and was carried out according to GLP.

Due to light sensitivity of test item, preparation of test solutions was performed under dimmed light conditions and glassware was wrapped in aluminium foil. Based on the very low water solubility of the test item, no toxicity to algae was expected and therefore a limit test at a nominal concentration of 5.0 µg/L was carried out.

The total exposure period was 72 hours and triplicate samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure, however no test item could be quantified in any of the samples taken from the test solutions. Therefore the limit test was repeated and additional samples taken from the stock solution to confirm the dosing concentration. The mean measured concentration in the 0.10 mg/mL stock solution was 0.094 mg/mL, indicating proper preparation of the stock and thus also test solutions. Therefore, effect parameters were expressed in terms of nominal concentrations.

In both limit tests, no significant differences were recorded between the values for growth rate or yield at the limit concentration when compared to the relevant control group(s).

A NOEC of 5 µg/L and an LC50 of >5 µg/L for both inhbition of growth and yield were determined. This concentration was above the limit of solubilty of the test substance.