Protein hydrolyzates, wool, reaction products with 3-chloro-2-hydroxypropyl-cocoalkyl-dimethylammonium chloride

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

From October 19, 2017 to January 05, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

not specified

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: Aerobic sludge, mixed treatment plant of urban (66%) and industrial (34%)

Details on inoculum:

Method of mud sampling
For the preparation of the inoculum a sample of aerobic sludge has been selected by the mixed treatment plant of urban (about 66%) and industrial (about 34%) liquid sewage situated at San Rocco - Monza (MB), Italia. The plant of treatment is managed by "BRIANZACQUE SRL (Monza)".

Mud preparation
In the laboratory the sampled muds, have been mixed and let settle, keeping them in aerobic conditions. The mud samples, before their use, have been analysed to check its ability to form colony units. Then it has been centrifuged, washed and analyzed to quantify the suspended solids concentration for the inoculum preparation. The inoculum was prepared in order to have a concentration of suspended solid of about 4 mg/L.

Medium preparation:

Solution A
Anhydrous potassium dihydrogen phosphate (KH2P04): 8.50 g/L
Anhydrous dipotassium hydrogen phosphate (K2HP04): 21.75 g/L
Disodium hydrogen phosphate dihydrate (Na2HP04.2H20): 33.40 g/L
Ammonium chloride (NH4Cl): 0.50 g/L
pH = 7.42

Solution B
Calcium chloride dihydrate (CaCI2.2H20): 36.40 g/L

Solution C
Magnesium sulphate heptahydrate (MgSO4.7H20): 22.50 g/L

Solution D
Iron chloride (Ill) hexahydrate (FeCl3.6H20): 0.25 g/L
All solutions has been made with deionized water (MilliQ Millipore). Then for each litre of medium 10 mL of Solution A have been transferred into a 1000 mL volumetric flask and diluted with 800 mL of deionised water; 1 mL of Solution B, 1 mL of Solution C and 1 mL of Solution D have been added and the solution has been diluted to volume with deionised water. For higher volumes the medium has been prepared according to the same proportion. The preparation of the solutions has been reported in an internal laboratory logbook.

Duration of test (contact time):

ca. 56 d

Initial conc.:

ca. 32.77 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Reference substance:

benzoic acid, sodium salt

Remarks:

23.70 mg/L of TOC

Key result

Parameter:

% degradation (inorg. C analysis)

Remarks:

Test substance

Value:

ca. 2

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Key result

Parameter:

% degradation (inorg. C analysis)

Remarks:

Test substance

Value:

ca. 7

Sampling time:

56 d

Remarks on result:

other: not readily biodegradable

Results with reference substance:

Percentage biodegradation of reference substance at 14 d was determined to be 74%, therefore the assay fulfilled the validity criteria.

Results

Quality criteria of the test were satisfied. The mean amount of TIC present in the blank controls at the end of the test was < 3 mg C/L. The trend of the inorganic carbon and related biodegradation percentages in the bottle contained both the test sample and the reference substance confirm the absence of inhibitory effect of the test sample on inoculum. The biodegradation percentage of the test substance during the test was as follow:

Check point (d)	% of Biodegradation of reference substance	% of Biodegradation of test substance	% of Biodegradation of reference substance + test substance
0	0	0	0
1	2	0	0
7	71	4	-
14	74	3	-
21	87	2	-
28	86	2	27
35	95	6	-
42	91	15	-
49	78	11	-
56	83	7	36

Conclusions

On the basis of results obtained, interpreted in accordance to OECD 310:2014, the test substance was considered not readily biodegradable in aerobic condition.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Based on results of the read across study, the test substance is considered as not readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance, 'Steardimonium hydroxypropyl hydrolysed wool' (active: 18.3%), using CO2 in sealed vessels (Headspace Test) method, according to OECD Guideline 310, in compliance with GLP. In the study, 29 replicates each were included for the inoculum blanks, positive control (sodium benzoate at 23.70 mgC/L) and read across substance (32.77 mgC/L) groups, whereas 11 replicates were included for the toxicity control (read across substance + reference substance) group. Aerobic sludge inoculum was prepared with mixed treatment plant of urban (66%) and industrial (34%) liquid sewage and in order to have a concentration of suspended solid of about 4 mg/L. Thereafter the inoculum was added in all bottles (except the inoculum blank), closed and incubated under stirring in darkness at 20°C for 56 d. The calculation of biodegradation was based on total inorganic carbon (TIC), which was determined using TOC analyser at each sampling time points i.e., on Day 0, 1, 7, 14, 21, 28, 35, 42, 49 and 56 for the reference substance, test sample and blank groups and on Day 1, 28 and 56 for the toxicity control group. The biodegradation percentage for the read across substance was determined to be 2% after 28 d and 7% after 56 d. As less than 60% degradation was achieved within 28 d, the read across substance was concluded to be not readily biodegradable under aerobic conditions. The trend of the inorganic carbon and related biodegradation percentages in the toxicity control group confirmed the absence of inhibitory effect of the read across sample on inoculum. After 14 d biodegradability for reference substance was determined to be 95%.The mean amount of TIC present in the blank controls at the end of the test was <3 mg C/L. Therefore, the test had met all the validity criteria. Under the study conditions, the read across substance was concluded to be not readily biodegradable (Eurofins, 2018).Based on results of the read across study, a similar biodegradation potential is expected for the test substance, Cocodimonium hydroxypropyl hydrolysed wool.

.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From October 10, 2017 to January 04, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

not specified

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: Aerobic sludge, mixed treatment plant of urban (66%) and industrial (34%)

Details on inoculum:

Method of mud sampling
For the preparation of the inoculum a sample of aerobic sludge has been selected by the mixed treatment plant of urban (about 66%) and industrial (about 34%) liquid sewage situated at San Rocco - Monza (MB), Italia. The plant of treatment is managed by "BRIANZACQUE SRL (Monza)".

Mud preparation
In the laboratory the sampled muds, have been mixed and let settle, keeping them in aerobic conditions. The mud samples, before their use, have been analysed to check its ability to form colony units. Then it has been centrifuged, washed and analyzed to quantify the suspended solids concentration for the inoculum preparation. The inoculum was prepared in order to have a concentration of suspended solid of about 4 mg/L.

Medium preparation:

Solution A
Anhydrous potassium dihydrogen phosphate (KH2P04): 8.50 g/L
Anhydrous dipotassium hydrogen phosphate (K2HP04): 21.75 g/L
Disodium hydrogen phosphate dihydrate (Na2HP04.2H20): 33.40 g/L
Ammonium chloride (NH4Cl): 0.50 g/L
pH = 7.47

Solution B
Calcium chloride dihydrate (CaCI2.2H20): 36.40 g/L

Solution C
Magnesium sulphate heptahydrate (MgSO4.7H20): 22.50 g/L

Solution D
Iron chloride (Ill) hexahydrate (FeCl3.6H20): 0.25 g/L
All solutions has been made with deionized water (MilliQ Millipore). Then for each litre of medium 10 mL of Solution A have been transferred into a 1000 mL volumetric flask and diluted with 800 mL of deionised water; 1 mL of Solution B, 1 mL of Solution C and 1 mL of Solution D have been added and the solution has been diluted to volume with deionised water. For higher volumes the medium has been prepared according to the same proportion. The preparation of the solutions has been reported in an internal laboratory logbook.

Duration of test (contact time):

ca. 56 d

Initial conc.:

ca. 26.19 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Reference substance:

benzoic acid, sodium salt

Remarks:

20.58 mg/L of TOC

Key result

Parameter:

% degradation (inorg. C analysis)

Remarks:

Test substance

Value:

ca. -1

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Key result

Parameter:

% degradation (inorg. C analysis)

Remarks:

Test substance

Value:

ca. 2

Sampling time:

56 d

Remarks on result:

other: not readily biodegradable

Results with reference substance:

Percentage biodegradation of reference substance at 14 d was determined to be 95%, therefore the assay fulfilled the validity criteria.

Results

Quality criteria of the test were satisfied. The mean amount of TIC present in the blank controls at the end of the test was < 3 mg C/L. The trend of the inorganic carbon and related biodegradation percentages in the bottle contained both the test sample and the reference substance confirm the absence of inhibitory effect of the test sample on inoculum. The biodegradation percentage of the test substance during the test was as follow:

Check point (d)	% of Biodegradation of reference substance	% of Biodegradation of test substance	% of Biodegradation of reference substance + test substance
0	0	0	0
1	3	5	0
7	87	1
14	95	2
21	99	10	-
28	110	-1	-1
35	93	1	-
42	94	0	-
49	97	5	-
56	96	2	1

Conclusions

On the basis of results obtained, interpreted in accordance to OECD 310:2014, the test substance was considered not readily biodegradable in aerobic condition.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Based on results of the read across study, the test substance is considered as not readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (active: 19.3%), using CO2 in sealed vessels (Headspace Test) method, according to OECD Guideline 310, in compliance with GLP. In the study, 29 replicates each were included for the inoculum blanks, positive control (sodium benzoate at 20.58 mgC/L) and read across substance (26.19 mgC/L) groups, whereas 11 replicates were included for the toxicity control (read across substance + reference substance) group. Aerobic sludge inoculum was prepared with mixed treatment plant of urban (66%) and industrial (34%) liquid sewage and in order to have a concentration of suspended solid of about 4 mg/L.Thereafter the inoculum was added in all bottles (except the inoculum blank), closed and incubated under stirring in darkness at 20°C for 56 d.The calculation of biodegradation was based on total inorganic carbon (TIC), which was determined using TOC analyser at each sampling time points i.e., on Day 0, 1, 7, 14, 21, 28, 35, 42, 49 and 56 for the reference substance, read across sample and blank groups and on Day 1, 28 and 56 for the toxicity control group. The biodegradationpercentage for the read across substance was determined to be -1% after 28 d and 2% after 56 d. As less than 60% degradation was achieved within 28 d, the read across substance was concluded to be not readily biodegradable under aerobic conditions.The trend of the inorganic carbon and related biodegradation percentages in the toxicity control group confirmed the inhibitory effect of the read across sample on inoculum. After 14 d biodegradability for reference substance was determined to be 95%.The mean amount of TIC present in the blank controls at the end of the test was <3 mg C/L. Therefore,the test had met all the validity criteria. Under the study conditions, the read across substance was concluded to be not readily biodegradable (Eurofins, 2018).Based on results of the read across study,a similar biodegradation potential is expected for the test substance,Cocodimonium hydroxypropyl hydrolysed wool.

Description of key information

Based on results of the read across studies, the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool', is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Study 1:

A study was conducted to determine the ready biodegradability of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (active: 19.3%), using CO2 in sealed vessels (Headspace Test) method, according to OECD Guideline 310, in compliance with GLP. In the study, 29 replicates each were included for the inoculum blanks, positive control (sodium benzoate at 20.58 mgC/L) and read across substance (26.19 mgC/L) groups, whereas 11 replicates were included for the toxicity control (read across substance + reference substance) group. Aerobic sludge inoculum was prepared with mixed treatment plant of urban (66%) and industrial (34%) liquid sewage and in order to have a concentration of suspended solid of about 4 mg/L.Thereafter the inoculum was added in all bottles (except the inoculum blank), closed and incubated under stirring in darkness at 20°C for 56 d.The calculation of biodegradation was based on total inorganic carbon (TIC), which was determined using TOC analyser at each sampling time points i.e., on Day 0, 1, 7, 14, 21, 28, 35, 42, 49 and 56 for the reference substance, read across sample and blank groups and on Day 1, 28 and 56 for the toxicity control group. The biodegradationpercentage for the read across substance was determined to be -1% after 28 d and 2% after 56 d. As less than 60% degradation was achieved within 28 d, the read across substance was concluded to be not readily biodegradable under aerobic conditions.The trend of the inorganic carbon and related biodegradation percentages in the toxicity control group confirmed the inhibitory effect of the read across sample on inoculum. After 14 d biodegradability for reference substance was determined to be 95%.The mean amount of TIC present in the blank controls at the end of the test was <3 mg C/L. Therefore,the test had met all the validity criteria. Under the study conditions, the read across substance was concluded to be not readily biodegradable (Eurofins, 2018).

Study 2:

A study was conducted to determine the ready biodegradability of the read across substance, 'Steardimonium hydroxypropyl hydrolysed wool' (active: 18.3%), using CO2 in sealed vessels (Headspace Test) method, according to OECD Guideline 310, in compliance with GLP. In the study, 29 replicates each were included for the inoculum blanks, positive control (sodium benzoate at 23.70 mgC/L) and read across substance (32.77 mgC/L) groups, whereas 11 replicates were included for the toxicity control (read across substance + reference substance) group. Aerobic sludge inoculum was prepared with mixed treatment plant of urban (66%) and industrial (34%) liquid sewage and in order to have a concentration of suspended solid of about 4 mg/L. Thereafter the inoculum was added in all bottles (except the inoculum blank), closed and incubated under stirring in darkness at 20°C for 56 d. The calculation of biodegradation was based on total inorganic carbon (TIC), which was determined using TOC analyser at each sampling time points i.e., on Day 0, 1, 7, 14, 21, 28, 35, 42, 49 and 56 for the reference substance, test sample and blank groups and on Day 1, 28 and 56 for the toxicity control group. The biodegradation percentage for the read across substance was determined to be 2% after 28 d and 7% after 56 d. As less than 60% degradation was achieved within 28 d, the read across substance was concluded to be not readily biodegradable under aerobic conditions. The trend of the inorganic carbon and related biodegradation percentages in the toxicity control group confirmed the absence of inhibitory effect of the read across sample on inoculum. After 14 d biodegradability for reference substance was determined to be 95%.The mean amount of TIC present in the blank controls at the end of the test was <3 mg C/L. Therefore, the test had met all the validity criteria. Under the study conditions, the read across substance was concluded to be not readily biodegradable (Eurofins, 2018).

The hydrolysed proteins differences across the read across substances is not expected to have an impact, as the hydrolysed proteins are known to be quickly degraded to more simple metabolites, called amino acids. Their persistence in the environment is very short, without any tendency to bioaccumulation. These compounds are present in live cells; consequently, they are not considered real waste, since they can be used again by the same live cells in the protein synthesis. Following to the evaluation of this Assessment Report, hydrolysed proteins were included in Annex I of Directive 91/414/EEC, as laid down in Commission Directive 2008/127/EC of 18 December 2008 and Commission Directive 2009/153/EC of 30 November 2009.

Therefore, based on the results of the read across studies, a similar biodegradation potential is expected for the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool'.

[Type of water: freshwater]