Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(dimethylamino)propyl]amino]sulfonyl derivs.

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Sep 2020 - 15 Sep 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Deviations:

no

Remarks:

A single and relatively higher concentration was used in order to increase the possibility of detecting and identifying metabolites which is the main goal of the study.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

- Location: Biggesee lake, 57462 Olpe-Sondern, North-RhineWestphalia, Germany
- Site description: A freshwater lake fed by a stream from a weir on the river. Wood- and grassland around the lake.
- Sampling date: September 08, 2020
- Sampling depth: 30-50 cm
- Procedure: Immersion of container
- Storage: 13 days at <= 8 °C
- Temperature (°C) at time of collection: 14.4°C
- pH at time of collection: 7.42
- Oxygen concentration at collection (mg/L): 9.51
- Oxygen concentration after filtration at date of application (mg/L): 8.4 / 8.0
- Dissolved organic carbon (mg/L): 1.8
- TOC water (mg/L): 1.58
- BOD (mg O2/L): 49.5
- Water filtered: yes

Duration of test (contact time):

61 d

Initial conc.:

1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

test mat. analysis

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 250 mL
- Solubilising agent: Ultra pure water + 0.02% formic acid
- Test temperature: 20 ± 2 °C
- pH: 7.84 - 8.27
- pH adjusted: no
-Redox potential [mV]: 171.6 - 225.5
- Aeration of dilution water: The samples were continuously saturated with humidified air (flow-through system).
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: The
incubation vessels and the round bottom flask used for sample work-up were rinsed by 10
mL DMSO for a second time.

TEST SYSTEM
- Culturing apparatus: 0.5 L Erlenmeyer flasks which were combined with three absorption traps of 100 mL for volatile degradation products
- Method used to create aerobic conditions: The samples were continuously saturated with humidified air (flow-through system)
- Method used to create anaerobic conditions: The sterilized samples were sealed during the incubation (no flow-through).

- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: yes except sterilized samples
- Details of trap for CO2 and volatile organics if used: The incubation flasks were combined with three absorption traps of 100 ml for volatile degradation products in series: ethylene glycol, 1 M
H2SO4 and 2 M sodium hydroxide solution.

SAMPLING
- Sampling frequency: Replicate samples were taken at 0 d (immediately after application), 7 d, 14 d, 28 d, 42 d and 61 d after application. Sterilised surface water samples were taken after 61 d.
- Sampling method used per analysis type: Whole flasks were sampled in duplicate at each sampling time.
- Sterility check if applicable: Yes. Measurement was conducted for a sterile sample alongside main samples
- Sample storage before analysis: After sampling, the vessels were opened, concentrated and worked-up immediately

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: not used
- Blank control: Untreated surface water samples
- Abiotic sterile control: For the preparation of the sterile (abiotic) samples, surface water and all required equipment was autoclaved at 121 °C for 20 minutes and the preparation of the samples was performed under a laminar flow workbench under sterile conditions.
- Reference control: 14C-sodium benzoate was added to the surface water to prepare the reference control
- Toxicity control: The toxicity control samples includes 14C-sodium benzoate ( 15.1 µg/L) as an easily degradable control substance and the test substance

Reference substance:

benzoic acid, sodium salt

Remarks:

14C labeled sodium benzoate was used

Test performance:

The concentration of parent radioactivity decreased from 95.4 % at day 0 to 73.2 % of the applied amount at the end of study period. However, as the signal system (retention time range 8-20 min) at final sampling time-point (61 d) contains both parent and metabolites due to incomplete separation of signal peaks, the amount of remaining parent test item cannot be exactly quantified.

Compartment:

natural water: freshwater

Sampling date:

2020

% Total extractable:

ca. 99

% CO2:

ca. 3.8

% Recovery:

96

St. dev.:

2.5

Remarks on result:

other: Percentage recovery is based on the mean of all samples collected for all sampling times

Parent/product:

parent

Compartment:

water

% Degr.:

22.2

Parameter:

radiochem. meas.

Sampling time:

61 d

Remarks on result:

other: Value calculated as the difference between initial (95.4 %) and final (73.2 %) measured radioactivity of the test substance using radio-HPLC

Parent/product:

parent

Compartment:

water

Key result

% Degr.:

> 50

Parameter:

radiochem. meas.

Sampling time:

61 d

Remarks on result:

other: Value based on semi-quantitative analysis by LC-HR-MS

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

No.:

#6

No.:

#7

Details on transformation products:

- General details: The structure of the test substance is complex and contains numerous variations basing on the copper phthalocyanine complex and only a part of these structures is identified. Furthermore, also the proposed structures are the most probable assumptions resulting from the determined chemical formula. Signals basing on the copper phthalocyanine complex not detected in blank sample and application solution will originate form metabolites formed during the incubation of the test substance in surface water. The determined chemical formula results from the evaluation of the mass spectroscopic measurement and is regarded as correct. However, the proposed structures are reasonable assumptions, but also alternatives are possible. For example, the loss of carbon atoms compared to the parent structure will be most probably caused by a stepwise degradation of the side chains. In case several side chains are present in the particular parent structure, different degradation processes may result in the same chemical formula and thus in the same detected masses. Such differences should lead to different retention times but regarding the complex nature of the test substance mixture, different retention times deliver only limited support for data interpretation.

- Formation and decline of each transformation product during test: Radio-HPLC analysis of the incubated samples showed two significant signals outside of the retention-time range of the test substance signal system that were increasing during the incubation time. The first of these signals, detected with maximal mean amounts of 4.4 % AR, eluted after approx. 0.5 - 1.5 min. At this retention time two masses and chemical formulas were detected with high intensities by the metabolite search. The chemical formulas are fitting to lead structure C representing species with side chains largely degraded to sulphonamides with formyl substituents. One of the species (No. 10.1/29) comprises four side chains, the other (No. 11.13/318) comprises three side chains and four hydroxyl substituents corresponding to a twofold dihydroxylation and re-aromatisaton. The second new signal, eluting after approx. 2.8 - 3.8 min, was detected with maximal mean amounts of 9.8 % AR. The metabolite screen delivered no results for this retention time range. The metabolite could be identified by a conventional search for reasonable structures like the detected phthalimide with a dimethylamino-propyl-aminosulfonyl side chain as present in the test substance. This metabolite results from the breakup of the phthalocyanine ligand system in the test substance and indicates that at least partially the core part of the phthalocyanine copper complex is degraded. As this metabolite was also found in the sterile sample, a chemical and non-microbial process may be responsible for the decomposition of the phthalocyanine copper complex.
A minor signal new signal (<1 % AR) was eluting after approx. 5 min. The mass detected in this retention time range resulted in a chemical formula fitting to a structure of component 4 after a twofold dihydroxylation (metabolite No. 42.3/464). The mass was detected at low-middle intensity corresponding to the low signal intensity in the radio channel and it was not detected in the application solution. As described above, assignment as dihydroxylation product is uncertain, but the chemical formula indicates an oxidation product of component 4. Further degradation products were found eluting within the retention time range of the test substance signal complex, confirming the assumption that degradation products are eluting together with the test substance. Metabolite No. 7/19 was eluting after 8.6 min and detected at high amounts. This metabolite belongs to lead structure B with three sulfonamide substituents and consequently represents a metabolite with mostly degraded side chains. Metabolite No. 21.1/127 was eluting after 6.4 min. and detected at medium amounts. This metabolite belongs to lead structure E with one partially degraded side chain. Finally, metabolite 41.2/459, dihydroxylated component 3, was found eluting after approx. 13.5 - 15-5 min and detected at middle-high amounts. Similar as for metabolite 42.3/464, not only a dihydroxlation but also another oxidation process may fit to the detected chemical formula.
Overall, the identified degradation products of indicate two different degradation processes. Oxidation reactions result in a successive degradation of the side chains of the test substance. The side chains are degraded to the corresponding sulphonamide moieties. Oxidation of the aromatic rings of the phthalocyanine ligands by dihydroxylation reactions is also possible. However, the core phthalocyanine copper complex is still present in the metabolites resulting from these oxidation processes. The identification of the substituted phthalimide as degradation product shows however, that also the phthalocyanine copper complex is at least partially degraded.


- Pathways for transformation: The identified degradation products of the test substance indicate two different degradation processes:
1) Oxidation reactions result in a successive degradation of the side chains of the test substance. The side chains are degraded to the corresponding sulphonamide moieties.
2) Oxidation of the aromatic rings of the phthalocyanine ligands by dihydroxylation reactions.

Evaporation of parent compound:

no

Remarks:

Only traces of radioactivity < 0.1 % of applied amount were detected in the absorption traps with ethylene glycol and 0.5M H2SO4.

Volatile metabolites:

no

Remarks:

The radioactivity detected in the absorption traps was almost completely found in the absorption trap with the sodium hydroxide solution for trapping 14CO2. Only < 0.1 % radioactivity was found in the other traps

Details on results:

TEST CONDITIONS
- Aerobicity, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered: Proper quantification and identification of remaining parent substance and transformated products was difficult due to a complex signal peak which comprises of both parent and metabolite substances.

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: 4.4 and 9.8 % on days 42 and 28, respectively
- Range of maximum concentrations in % of the applied amount at end of study period: 3.8 and 7.9 %

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 3.8

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: < 0.1

STERILE TREATMENTS
- Transformation of the parent compound: In the samples incubated at sterile conditions the metabolites observed at non-sterile conditions were also detected, but at significantly lower amounts

Results with reference substance:

About 50% of the applied 14C-sodium benzoate were mineralised after an incubation time of
approx. 5 d. At the end of the incubation period (26 d) more than 79 % of the applied
radioactivity was mineralised in all reference control samples. This indicates an appropriate
biological activity in the surface water tested.

A toxicity sample was also measured to exclude that the high concentration of the test substance induced adverse effect on microbial activity.
In the toxicity sample, 50 % of the applied 14C-sodium benzoate were mineralised after an incubation time of approx. 7 d.
At the end of the incubation period (26 d) more than 68 % of the applied radioactivity was mineralised in all reference control samples.

Pretest with non-labelled Test substance
Solubility (non-GLP): The aqueous stock solution prepared by Fraunhofer IME at a concentration of 0.375 mg/mL was controlled optically and no suspended particles were observed. Samples prepared and analysed by Currenta GmbH with concentrations of 1 mg/L, 10 mg/L and 100 mg/L delivered stable detector responses measured at 0, 1 and 4 days after preparation, showing that within this concentation range solutions of the test substance in acidified water are stable. Depending on the particular sample and analysed species, relative standard deviations were between 1 % and 17%.

Pretest with 14C labelled Test substance
Qptimization of work-up method for surface water samples: Samples of natural surface water (250 ml) were applied with 14C-test substance at a concentration of 1 mg/L. The exact amount of radioactivity was determined by Liquid scintillation counting. The liquid phase was removed by rotary evaporation at 60 °C and 100 mbar to dryness. The residue was redissolved in a 5 mL DMSO. The recoveries for a sample duplicate were determined by Liquid scintillation to be 94.6 % and 94.5 %. The resulting solution could be directly analysed by radio-HPLC with injection volumes up to 10 µL.

 

Distribution of radioactivity to compartments and mass balance
The mass balance and the distribution of the applied radioactivity during the aerobic mineralization of 14C-test substance in surface water is presented in Table 1. The mass balance for the individual samples was calculated from the radioactivity in the DMSO solutions after sample work-up and the radioactivity in the sodium hydroxide trap.

Table 1: Mass balance in surface water samples treated with test substance at a concentration of 1 mg/L and incubated at 20 °C in % of applied radioactivity

	% of Applied Radioactivity by lncubation time (days)
	Replicate	0	7	14	28	42	61	sterile 61
DMSO solution	1	99.0	94.3	96.5	89.0	95.3	84.9	93.1
	2	99.0	97.0	96.8	94.1	92.3	91.7	91.8
	mean	99.0	95.7	96.6	91.6	93.8	88.3	92.5
Volatile Traps*	1	0.0	0.6	1.0	2.7	3.4	4.5	no sample
	2	0.0	0.6	1.0	2.6	3.1	3.1	no sample
	mean	0.0	0.6	1.0	2.7	3.2	3.8	no sample
Total	1	99.0	94.9	97.5	91.7	98.7	89.4	93.1
	2	99.0	97.6	97.8	96.8	95.4	94.8	91.8
	mean	99.0	96.3	97.7	94.2	97.0	92.1	92.5
Overall Mean ± StdDev	96.0 ± 2.5

* Corresponds to radioactivity in 2 N NaOH solution. Radioactivity in ethylene glycol and 0.5 M H2SO4 < 0.1 % of applied amount

 

ldentification of extractable radioactivity

Formation of metabolites showed the degradation of the test substance in surface water. During 61 days of incubation, the parent substance was degraded to several transformation products, which were characterised by their retention time during HPLC analysis, as no reference standards were available. By this way, one unknown transformation product was detected exceeding four times 5 % of applied radioactivity (AR) which was specified as unassigned metabolite with the retention time of 3.3 min.

Due to the nature of the test substance as complex mixture of components with similar chromatographical properties covering a wide range of polarity, the analysis of the chromatographical results is limited or not distinct. Same metabolisation steps on different species within the parent mixture will lead to mixtures with similar chromatographical properties. Consequently, the resulting signals of metabolites are expected to consist of several species. Furthermore, the broad signal system of the parent mixture (retention time rage 8-20 min) overlays signals originating from metabolites. Consequently, the signal system (retention time rage 8-20 min) contains an increasing amount of metabolites with increasing incubation time.

The amount of the metabolite eluting with the retention time of 1.1 min, the second highest metabolite observed, increased to maximal mean value of 4.4 % AR after 42 days of incubation and were detected at mean value of 3.8 % AR after 61 days of incubation. The corresponding signal was already observed in the application solution at amounts of approx. 1.5 % AR but the amount clearly increased during the incubation time.

The amount of the main metabolite eluting with the retention time of 3.3 min increased to maximal mean value of 9.8 % AR after 28 days of incubation and was detected at mean value of 7.9 % AR after 61 days of incubation. Starting with samples taken at incubation day 14, the amount exceeded 5 % AR. The metabolite was detected with a mean amount of 1.3 % AR also in the 0 d samples, worked-up immediately after application. Therefore, it cannot be excluded that this amount was generated during the work-up process. However significantly increased amounts for later incubation times clearly show, that it was mainly formed during the incubation process.

The signal detected at a retention time of 33.3 min was observed at amount <1 % AR in the non sterile samples and 1.4 % AR in the sterile samples. Due to the low amount it is not fully clear if the amount is systematically increasing during the incubation. In the subsequent analyses by HR-MS-ToF at this retention time, several masses were observed at low intensity that were also detected at significantly lower retention times but partially much higher intensity. Some of these masses were also detected in blank samples (solvent only). Considering these results, it is assumed that the occurrence of this signal is a chromatographical error. lt seems that a mixture of residual compounds was not completely eluted from the column or remained in the LC-machine and was eluted together at the end of the gradient forming the observed signal. Therefore, the signal observed at 33.3 min is not regarded as an independent metabolite.

Further five minor metabolites eluting with the retention times of 4.3 min, 5.3 min, 6.0 min, 7.2 min and 7.9 min were observed after an incubation time of 14 d and later. The mean amounts of these metabolites were below 1 % AR for sampling times, except for the metabolite eluting with a retention time of 6.0 min, which was observed at an amount of 1.1 % AR after 42 d of incubation.
In the samples incubated at sterile conditions, the metabolites observed at non-sterile conditions were also detected, but at significantly lower amounts. Approx. half of the amount was observed for the metabolites eluting with the retention times of 1.1 and 3.3 min (2.0 % and 4.0 % AR) when compared to corresponding sample incubated for 61 d at non-sterile conditions. Five further, minor metabolites were observed at mean amounts not exceeding 0.2 % AR. The result of the sterile sample indicates that the degradation of the test substance was at least partially a chemical process without microbial contribution. The detected 14CO2 in the sodium hydroxide traps proves degradation by microbial activity therefore the degradation of the test substance may also be the result of a combined microbial and chemical process.

All of the detected metabolites eluted at shorter retention times compared to Astra Blaubase 6GLL. This indicates the formation of species with higher polarity compared to the parent test item.

The results presented in Table 2 show the amounts of parent and its metabolites determined in DMSO solution after sample work-up.
Determinations are based on radio-HPLC analysis of the DMSO solution. The values are expressed in percent of the total initially applied radioactivity (AR). Radio-HPLC was chosen as primary (quantitative} analytical method.

Table 2: Pattern of parent test substance and its metabolites in surface water treated at concentratlon of 1 mg/L determined in DMSO solution by radio-HPLC.

Substance	Replicate	% of Applied Radioactivity by Sampling times (days)
		0	7	14	28	42	61	61 sterile
Signal RT 1.1 min	1	2.3	1.5	3.0	3.6	4.6	4.0	2.3
	2	2.2	1.6	2.9	3.4	4.2	3.7	1.8
	mean	2.3	1.6	2.9	3.5	4.4	3.8	2.0
Signal RT 3.3 min	1	1.5	0.9	8.1	10.2	7.2	8.9	3.7
	2	1.1	1.2	9.0	9.3	7.9	6.8	4.3
	mean	1.3	1.1	8.5	9.8	7.6	7.9	4.0
Signal RT 4.3 min	1	0.0	0.0	0.1	0.5	0.6	0.6	0.1
	2	0.0	0.0	0.6	0.5	0.3	0.7	0.1
	mean	0.0	0.0	0.3	0.5	0.5	0.7	0.1
Signal RT 5.3 min	1	0.0	0.0	0.3	0.0	0.0	0.7	0.2
	2	0.0	0.0	0.3	0.3	0.0	0.3	0.2
	mean	0.0	0.0	0.3	0.1	0.0	0.5	0.2
Signal RT 6.0 min	1	0.0	0.0	0.5	0.9	1.0	0.5	0.1
	2	0.0	0.1	0.8	0.7	1.1	0.6	0.3
	mean	0.0	0.0	0.6	0.8	1.1	0.5	0.2
Signal RT 7.2 min	1	0.0	0.0	0.6	0.7	1.0	1.3	0.1
	2	0.0	0.0	0.5	0.4	0.7	0.3	0.1
	mean	0.0	0.0	0.5	0.6	0.8	0.8	0.1
Signal RT 7.9 min	1	0.0	0.0	0.0	0.2	0.3	0.0	0.0
	2	0.0	0.0	0.0	0.3	0.3	0.1	0.2
	mean	0.0	0.0	0.0	0.2	0.2	0.1	0.1
Parent RT 8-20 min	1	95.2	91.5	84.1	72.5	80.3	68.2	85.1
	2	95.7	93.8	82.7	78.8	77.7	78.3	83.6
	mean	95.4	92.6	83.4	75.6	79.0	73.2	84.3
Signal RT 33.5 min	1	0.0	0.4	0.0	0.3	0.3	0.8	1.5
	2	0.0	0.3	0.0	0.4	0.1	0.8	1.2
	mean	0.0	0.3	0.0	0.4	0.2	0.8	1.4

Validity criteria:

The reference substance is expected to degrade in less than 2 weeks

Observed value:

About 50% of the applied 14C-sodium benzoate were mineralised after an incubation time of
approx. 5 d. At the end of the incubation period (26 d) more than 79 % of the applied
radioactivity was mineralised in all reference control samples. This indicat

Validity criteria fulfilled:

yes

Validity criteria:

The total recovery (mass balance) at the end of the experiment should be between 90% and 110%
for radiolabelled substances

Observed value:

96 ± 2.5

Validity criteria fulfilled:

yes

Conclusions:

Degradation of the test substance was observed during incubation in surface water. Reliable quantification of the degradation is in most cases not possible as parent and metabolites are partially not separated by HPLC and eluting within the same retention time range. Seven metabolites could be identified by structure. Semi-quantitative analysis by LC-HR-MS indicate degradation rates >50% for the observed components of the test substance. The identification of degradation products is highly complex due to the large number of possible metabolites and the chromatographic behaviour of the resulting mixture. The results however indicate the oxidative degradation of the side chains as well as the complete breakup of the phthalocyanine copper complex.

Executive summary:

The degradation and biotransformation of the test substance was studied in filtered surface water (pH 7.42, dissolved organic carbon 1.8 mg/L) from Biggesee lake, 57462 Olpe-Sondern, North-Rhine Westphalia, Germany for 61 d under aerobic conditions in dark at 20 ± 2 ºC. The test substance was radio-labeled and applied at the rate of 1 mg/L. The experiment was conducted in accordance with the OECD guideline 309, and in compliance with GLP standards. The study design was adopted to special requirements to focus on the detection, characterization and if possible the identification of degradation products, while determination of DT50 values and degradation kinetics was not the focus. Consequently, a single high concentration of the test substance of 1 mg/L was chosen to ensure sufficient analytical sensitivity regarding the complex nature of the substance.

 

The test system consisted of 0.5 L Erlenmeyer flasks which were combined with three absorption traps of 100 mL for volatile degradation products. Samples were analysed at 0 d (immediately after application), 7 d, 14 d, 28 d, 42 d and 61 d after application by Liquid scintillation counting and High performance liquid chromatography (HPLC). The water samples were evaporated to dryness and redissolved in DMSO, and the test substance residues were analysed by HPLC–UV. Identification of the transformation products was done by HPLC with both radio-detection and Mass spectrometry (MS-ToF). Radioactive mass balance was 96 ± 2.5 % of the applied amount.

 

The results show some mineralisation (approx. 4.0 %) of the test substance during the aerobic incubation for up to 61 d and degradation was observed by the formation of metabolites. Two new signals, reaching maximal amounts of 4.4 % and 9.8 % of the applied amount were eluting with shorter retention times compared to the parent substance. The metabolite observed at 9.8 % after 28 d of incubation was identified to originate from breakup of the phthalocyanine ligand system resulting in a phthalimide structure with intact side chain. The metabolite observed at 4.4 % after 42 d of incubation and further metabolites identified within the broad signal system of parent, were products of a successive oxidative degradation of the side chains of the test substance and/or dihydroxylation at the phenyl group. The quantity of these metabolites could not be determined due to incomplete separation of parent and metabolites leading to elution within the same retention time range. The identification of metabolites was performed by searching HR-MS-ToF data of 14C of parent substance and an incubated pooled sample for a set of 470 potential metabolite masses. In total 7 metabolites could be identified and structures are proposed. The concentration of parent radioactivity decreased from 95.4 % at day 0 to 73.2 % of the applied amount at the end of study period resulting in an estimated degradation of 22.2 %. However, as the signal system (retention time range 8-20 min) at final sampling time-point (61 d) contains both parent and metabolites, the amount of remaining parent test item cannot be exactly quantified. Therefore, a semi-quantitative comparison of HR-MS signal intensities of selected main species of parent 14C was performed to give a better estimate of the percentage degradation. This semi-quantitative analysis indicates that < 50 % of the initial parent amount is present at the end of incubation leading to percentage degradation of > 50 %. Although the value of > 50% is semi-quantitative but it is believed to better describe the degradation behaviour.  

 

Sterilised samples were used to check for abiotic degradation. The same metabolites eluting with shorter retention times as in the non-sterile samples were observed, but at significantly lower amounts. The amount of radioactivity within the broad signal system of parent in sterile samples was found to be 84.3 % of the applied amount at the end of the incubation period of 61 days. The result of the sterile sample indicates that the degradation of the test substance was at least partially a chemical process without microbial contribution. The detected 14CO2 proves degradation by microbial activity.

 

In conclusion, the > 50 % degradation (estimated based on semi-quantitative analysis) of the test substance may be the result of a combined microbial and chemical process leading to the oxidative degradation of the side chains (oxidative de-amination) as well as the complete breakup of the phthalocyanine copper complex following dihydroxylation of the aromatic ring.

Description of key information

The degradation and biotransformation of the test substance was studied in natural surface water from Biggesee lake, 57462 Olpe-Sondern, North-Rhine Westphalia, Germany for 61 d under aerobic conditions in dark at 20 ± 2 ºC. The test substance was radio-labeled and applied at the rate of 1 mg/L. The experiment was conducted in accordance with the OECD guideline 309, and in compliance with GLP standards. The study design was adopted to special requirements to focus on the detection, characterization and if possible the identification of degradation products, while determination of DT50 values and degradation kinetics was not the focus.
The test substance was incubated with the surface water and samples were analysed at 0 d (immediately after application), 7 d, 14 d, 28 d, 42 d and 61 d after application. The results show that degradation of the test substance was observed during incubation in surface water. Reliable quantification of the degradation is in most cases not possible as parent and metabolites are partially not separated by HPLC and eluting within the same retention time range. Seven metabolites could be identified by structure. Semi-quantitative analysis by LC-HR-MS indicates degradation rates >50% for the observed components of the test substance.
In conclusion, the > 50 % degradation (estimated based on semi-quantitative analysis) of the test substance may be the result of a combined microbial and chemical process leading to the oxidative degradation of the side chains (oxidative de-amination) as well as the complete breakup of the phthalocyanine copper complex following dihydroxylation of the aromatic ring.

In addition, the biodegradation of the test substance in sediment has been waived due to the low adsorption potential of the substance (log Koc = 0.96) according to REACH regulation, annex IX, column 1. Therefore, a sediment simulation study is not required.