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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th June 20 - 25th October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

version 2004

Deviations:

no

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

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under storage conditions: stable at room temperature protected from the light
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Very low water solubility.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1 test concentration, 5.0µg/l
- Sampling method: 1.0ml taken from the approximate centre of the test vessels
- Sample storage conditions before analysis: samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
- Other information: at the end of the exposure period, the replicates were pooled at each concentration before sampling. In addition, 1.0ml triplicates samples were taken from stock solutions in THF used in the preparation of test solutions.

Test solutions

Vehicle:

yes

Remarks:

tetrahydrofuran (THF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with dissolving test item in tetrahydrofuran (THF) to obtain a stock solution with a nominal concentration of 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. Test solutions were magnetically stirred for 15 miutes to assure homogenous test solutions.
- Controls: Test medium without test item or other additives (blank control) and test medium without test item but with solvent used in the treatment, 25µl blank THF (solvent control).
- 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.
- Other relevant information: preparation of test solutions was performed under dimmed light conditions and glassware was wrapped in aluminium foil to minimise exposure to light.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: not stated
- Age at study initiation (mean and range, SD): at least third generation, obtained by cyclical parthenogenesis under specified breeding conditions. Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.
- Method of breeding: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all glass culture vessel. Cultures were renewed after 7 days by renewing half of the medium twice a week. Cultured at 18 to 22°C, in an M7 suspension as prescribed in Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33.
- Source: In house laboratory culture with a known history
- Age of parental stock (mean and range, SD):
- Feeding during test : not fed during test

ACCLIMATION : no acclimation period detailed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

CaCO3 180mg/l

Test temperature:

19 to 20°C

pH:

7.8 to 8.2

Dissolved oxygen:

8.8 to 9.0

Salinity:

not stated

Conductivity:

not stated

Nominal and measured concentrations:

Nominal: 5.0µg/l test concentration, considered approximately 5 times above the limit of solubility.
Measured: Samples taken from the 10µg/l stock solution had a recovery of 37, 24 and 50%. From this an exposure concentration in the test solutions of 3.8µg/l was determined.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60ml, all glass
- Type (delete if not applicable): not stated
- Material, size, headspace, fill volume: glass, 10ml headspace
- Volume of solution: 50ml of solution
- Aeration: no aeration
- No. of organisms per vessel: 5 per vessel
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates
- No. of vessels per vehicle control (replicates): 4 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/l

OTHER TEST CONDITIONS
- Adjustment of pH: not stated
- Photoperiod: study performed in the dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility at 24 and 48 hours

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY : NO

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 3.8 µg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: concentration based on measured stock solution concentration

Details on results:

The 48h-EC50 for Daphnia magna exposed to 2,9,16,23-tetrakis(2,5-di-tert- butyl-4-methoxyphenoxy)phthalocyanine was beyond the average exposure concentration of 3.8 µg/L, which was above the maximum water solubility limit of 1.0 µg/L.

Results with reference substance (positive control):

- Results with reference substance valid? - valid
- Limit test: No
- Dose-response test: range tested between 0.10 and 1.8 mg/l
- ECx: 48 hour EC50 0.55 mg/l with a 95% confidence interval between 0.47 and 0.64 mg/l. The 48h-EC50 was within the expected range of 0.28 and 0.75 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.

Any other information on results incl. tables

Table 1. Number of introduced daphnids and incidence of immobility in the limit test

Time	Replicate	Average concentration (µg/l)
		Blank control	Solvent control	3.8
0	A	5	5	5
	B	5	5	5
	C	5	5	5
	D	5	5	5
	Total introduced	20	20	20
24	A	0	0	0
	B	0	0	0
	C	0	0	0
	D	0*	0	0
	Total immobilised	0	0	0
	Effect %	0	0	0
48	A	0	0	0
	B	0	0	0
	C	0	0	0
	D	0	0	0
	Total immobilised	0	0	0
	Effect %	0	0	0

* One daphnid was observed to be trapped at the surface of the test solution. This daphnid was reimmersed into solution before recording of mobility.

Table 2. Limit test: the test sample results (12th June 2019)

Time ofsampling[hours]	Concentration [µg/L]			Relativeto  nominal[%]	Mean relative to nominal (CV) [%]	Relativeto  initial [%]	Mean relativetoinitial [%]
	Nominal	Analyzed	Mean Analyzed
0	0	<1.01	<1.01	n.a.	n.a.
	0	<1.01		n.a.	(n.a.)
	0	<1.01		n.a.
	0	<1.01		n.a.
	0	<1.01		n.a.
	0	<1.01	<1.01	n.a.	n.a.
	0	<1.01		n.a.	(n.a.)
	0	<1.01		n.a.
	0	<1.01		n.a.
	0	<1.01		n.a.
	0	<1.01	<1.01	n.a.	n.a.
	0	<1.01		n.a.	(n.a.)
	0	<1.01		n.a.
	0	<1.01		n.a.
	0	<1.01		n.a.
	0-THF	<1.01	<1.01	n.a.	n.a.
	0-THF	<1.01		n.a.	(n.a.)
	0-THF	<1.01		n.a.
	0-THF	<1.01		n.a.
	0-THF	<1.01		n.a.
	0-THF	<1.01	<1.01	n.a.	n.a.
	0-THF	<1.01		n.a.	(n.a.)
	0-THF	<1.01		n.a.
	0-THF	<1.01		n.a.
	0-THF	<1.01		n.a.
	0-THF	<1.01	<1.01	n.a.	n.a.
	0-THF	<1.01		n.a.	(n.a.)
	0-THF	<1.01		n.a.
	0-THF	<1.01		n.a.
	0-THF	<1.01		n.a.
	5.0	2.96	2.96	59	59
	5.0	2.99		60	(1.8)
	5.0	2.93		59
	5.0	3.01		60
	5.0	2.88		58
	5.0	2.84	2.84	57	57
	5.0	2.76		55	(1.6)
	5.0	2.89		58
	5.0	2.87		57
	5.0	2.85		57
	5.0	2.50	1.92	50	38
	5.0	1.57		31	(23)
	5.0	2.27		45
	5.0	1.45		29
	5.0	1.83		37

1         Any response measured in the samples was below the concentration of the lowest calibration solution ofthe bracket (i.e. 0.50 µg/L). The concentration was reported <1.0 µg/L taking the dilution factor of 2 into account. The maximum contribution to the lowest sample concentration is 11% taking the dilution factor into account.

#         Date of sampling andanalysis.

n.a. Not applicable.

Table 3. Limit test: the test sample results (14th June 2019#)

Time ofsampling[hours]	Concentration [µg/L]			Relativeto  nominal[%]	Mean relative to nominal (CV) [%]	Relativeto  initial [%]	Mean relativetoinitial [%]
	Nominal	Analyzed	Mean Analyzed
48	0	<1.01	<1.01	n.a.	n.a.	n.a.	n.a.
	0	<1.01		n.a.	(n.a.)	n.a.
	0	<1.01		n.a.		n.a.
	0	<1.01		n.a.		n.a.
	0	<1.01		n.a.		n.a.
	0	<1.01	<1.01	n.a.	n.a.	n.a.	n.a.
	0	<1.01		n.a.	(n.a.)	n.a.
	0	<1.01		n.a.		n.a.
	0	<1.01		n.a.		n.a.
	0	<1.01		n.a.		n.a.
	0	<1.01	<1.01	n.a.	n.a.	n.a.	n.a.
	0	<1.01		n.a.	(n.a.)	n.a.
	0	<1.01		n.a.		n.a.
	0	<1.01		n.a.		n.a.
	0	<1.01		n.a.		n.a.
	0-THF	<1.01	<1.01	n.a.	n.a.	n.a.	n.a.
	0-THF	<1.01		n.a.	(n.a.)	n.a.
	0-THF	<1.01		n.a.		n.a.
	0-THF	<1.01		n.a.		n.a.
	0-THF	<1.01		n.a.		n.a.
	0-THF	<1.01	<1.01	n.a.	n.a.	n.a.	n.a.
	0-THF	<1.01		n.a.	(n.a.)	n.a.
	0-THF	<1.01		n.a.		n.a.
	0-THF	<1.01		n.a.		n.a.
	0-THF	<1.01		n.a.		n.a.
	0-THF	<1.01	<1.01	n.a.	n.a.	n.a.	n.a.
	0-THF	<1.01		n.a.	(n.a.)	n.a.
	0-THF	<1.01		n.a.		n.a.
	0-THF	<1.01		n.a.		n.a.
	0-THF	<1.01		n.a.		n.a.
	5.0	5.61	5.59	112	112	189	189
	5.0	5.94		119	(7.6)	198
	5.0	5.96		119		203
	5.0	5.52		110		183
	5.0	4.91		98		171
	5.0	6.48	6.16	130	123	228	217
	5.0	6.03		121	(3.9)	218
	5.0	6.20		124		215
	5.0	5.84		117		204
	5.0	6.27		125		220
	5.0	4.31	5.02	86	100	173	275
	5.0	5.34		107	(9.0)	341
	5.0	5.45		109		240
	5.0	5.12		102		354
	5.0	4.89		98		267

1         Any response measured in the samples was below the concentration of the lowest calibration solution ofthe bracket (i.e. 0.50 µg/L). The concentration was reported as <1.0 µg/L taking the dilution factor of 2 into account. The maximum contribution to the lowest sample concentration is 15% taking the dilution factor into account.

#         Date of sampling andanalysis.

n.a. Not applicable.

Table 4. Stock solution sample results

Time ofsampling[hours]	Date ofsampling	Date of analysis1	Concentration [mg/mL]			Relativeto  nominal[%]	Mean relative to nominal (CV) [%]
			Nominal	Analyzed	Mean Analyzed
0	12 Jun 2019	12 Jun 2019	0-THF	<5x10-7	<5x10-7 (1)	n.a.	n.a.
			0-THF	<5x10-7		n.a.	(n.a.)
			0-THF	<5x10-7		n.a.
			0-THF	<5x10-7		n.a.
			0-THF	<5x10-7		n.a.
			0.10	0.0382	0.037	38	37
			0.10	0.0352		35	(11)
			0.10	0.0372		37
			0.10	0.0422		42
			0.10	0.0312		31
			0-THF	<5x10-7	<5x10-7 (1)	n.a.	n.a.
			0-THF	<5x10-7		n.a.	(n.a.)
			0-THF	<5x10-7		n.a.
			0-THF	<5x10-7		n.a.
			0-THF	<5x10-7		n.a.
			0.10	0.0392	0.024	39	24
			0.10	0.0262		26	(45)
			0.10	0.0162		16
			0.10	0.0112		11
			0.10	0.0262		26
			0-THF	<5x10-7	<5x10-7 (1)	n.a.	n.a.
			0-THF	<5x10-7		n.a.	(n.a.)
			0-THF	<5x10-7		n.a.
			0-THF	<5x10-7		n.a.
			0-THF	<5x10-7		n.a.
			0.10	0.0510	0.050	51	50
			0.10	0.0482		48	(7.7)
			0.10	0.0462		46
			0.10	0.0502		50
			0.10	0.0560		56

1         Any response measured in the samples was below the concentration of the lowest calibration solution ofthe bracket (i.e. 0.50 µg/L). The concentration was reported as <5x10-7mg/mL.

2         Estimated value, calculated by extrapolation of the calibrationcurve.

n.a. Not applicable.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 for Daphnia magna exposed to 2,9,16,23-tetrakis(2,5-di-tert- butyl-4-methoxyphenoxy)phthalocyanine was beyond the average exposure concentration of 3.8 µg/L, which was above the maximum water solubility limit of 1.0 µg/L. EC50> 3.8µg/L

Executive summary:

The study evaluated 2,9,16,23-tetrakis(2,5-di-tert-butyl-4- methoxyphenoxy)phthalocyanine for its ability to generate acute toxic effects on the mobility of Daphnia magna over an exposure period of 48 hours. The study was carried out according to the OECD 202 guideline and in accordance with GLP. Preparation of test solutions was performed under dimmed light conditions and glassware was wrapped in aluminium foil to minimize exposure to light. Since the test item is poorly soluble in water, a limit test was performed exposing daphnids to a nominal concentration of 5µg/l which was above the maximum solubility limit. Twenty daphnids per group (4 replicates) were exposed to a blank control, solvent control and to a nominal concentration of 5.0 µg/L. Analytical determination of the exposure concentration determined an average exposure concentration of 3.8 µg/L. Triplicate samples were also taken from the stock solution in THF were analysed in 5-fold. The mean measured concentrations were at 24, 37 and 50% relative to the nominal concentration of 0.10 mg/mL. No immobility was observed in the controls and at the limit concentration throughout the test. In conclusion, the 48h-EC50 for Daphnia magna exposed to 2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine was beyond the average exposure concentration of 3.8 µg/L, which was above the maximum water solubility limit of 1.0 µg/L.