Alcohols, C20-22, reaction products with phosphorus oxide (P2O5)

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 October 2019 - 10 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Specific details on test material used for the study:

Physical State/Appearance: White flakes powder
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Samples of the fresh test preparations were taken on Days 0, 6, 13 and 20 and of the expired test preparations on Days 1, 7, 14 and 21 Samples were taken for immediate analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Nominal amounts of test item were each separately added to the surface of test water to give the required loading rates. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid depth siphoning (the first approximate 75 to 100 mL discarded) to give the required loading rate WAFs.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 mL glass vessels containing Elendt M4 medium in a temperature controlled room at approximately 20 ºC. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata (formally known as Pseudokirchneriella subcapitata)) and GEMMA Micro 300 fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Remarks on exposure duration:

Daphnia magna were exposed to a WAF of the test item over a range of test concentrations of for a period of 21 days.

Post exposure observation period:

n/a

Hardness:

242 to 268 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.

Test temperature:

Temperature was maintained at approximately 19 ºC to 22 ºC throughout the test.

pH:

7.9 - 8.6

Dissolved oxygen:

8.0 - 9.5 mg O2/L

Salinity:

CaCl2.2H2O - 293.8mg/L
KCl - 5.8mg/L

Conductivity:

n/a

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/L.
Based on the results of a preliminary range finding test, Daphnia magna were exposed to a WAF of the test item over a range of test concentrations of 10, 18, 32, 56 and 100 mg/L for a period of 21 days.

Details on test conditions:

For each concentration a single daphnid was placed in 100 mL of the test preparation in 120 mL glass vessels which were then covered to reduce evaporation. For each test and control group 10 replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 ºC with a maximum deviation of ±1 ºC with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed daily throughout the test. The adult Daphnia were transferred to fresh media by wide bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
Each daphnid received approximately 5 to 20 µL of an algal suspension (Raphidocelis subcapitata) daily and approximately 20 µL of GEMMA Micro 300 fish food suspension on Days 0 to 3. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

morphology

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

morphology

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

morphology

Details on results:

Range-finding Test:
No immobilization was observed at the test concentrations of control, 10 and 100 mg/L loading rate WAFs. No sub-lethal effects of exposure was observed in all test concentrations
Chemical analysis of the fresh test preparations on Day 0 showed measured test concentrations of 0.00036 to 0.0012 mg/L. The chemical analysis of the old test preparations on Day 2 showed measured test concentrations to range from less than the limit of quantification (LOQ) to 0.00010 mg/L, showing that the test item appears to be unstable under the test conditions.

Definitive Test:
Chemical analysis of the fresh test preparations on Days 0, 6, 13 and 20 showed measured test concentrations to range from less than the limit of quantification (LOQ), which was determined to be 0.00074 to 0.058 mg/L. Chemical analysis of the aged test preparations on Days 1, 7, 14 and 21 showed measured test concentrations to range from less than the LOQ to 0.054 mg/L.

Lethal Effects on the Parental Generation (P1):
One mortality was observed at the test loading rate of 10 mg/L on Day 7. However, statistical analysis of the mortality data using the Fisher’s Exact Binomial Test with Bonferroni Correction test showed that the observed mortality in the 10 mg/L loading rate WAF group were not significantly different (P0.05) when compared to the control group.
No mortalities occurred at the control, 18, 32, 56 and 100 mg/L loading rate WAF groups throughout the test.

Sub-lethal Effects on the Parental Generation (P1):
The daphnids in all test concentrations were observed to be the same size and color as the control daphnids apart from one daphnid in the 10 mg/L loading rate WAF test group which was observed to be pale on Day 6 before being observed to be dead on Day 7.
The results showed that there were no statistically significant differences (P0.05) between the control and the 10, 18 and 32 mg/L loading rate WAF test groups in terms of length of the daphnids after 21 days exposure to the test item. The 56 and 100 mg/L loading rate WAF test groups showed a statistically significant difference from the control after 21 days in terms of length of the daphnids.

Effects on Reproduction:
After 21 days there were no statistically significant differences between the control and all the test groups in terms of the number of live young produced per adult.

Effects on the Filial Generation (F1):
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.
Young were first produced in the control test group on Day 9 of the test.

Water Quality Criteria:
Temperature was maintained at approximately 19 ºC to 22 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The water hardness was observed to be in the range 242 to 268 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.
Throughout the test the light intensity was observed to be in the range 892 to 1426 lux.

See tables 1 - 6.

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

The 21 Day ELx values for parental generation immobilization were calculated by Probit analysis using Linear Maximum-Likelihood Regression.
The ELx values for reproduction after 21 days were calculated by Probit analysis using Linear Maximum-Likelihood Regression.
The ELx values for body length were calculated using the 3-Parameter Normal Cumulative Distribution Function.
All ELx determinations were performed using the ToxRatPro software package (TOXRAT).

For the determination of the Lowest Observed Effect Loading Rate (LOEL) and the No Observed Effect Loading Rate (NOEL), the number of parental generation Daphnia mortalities for the control and each test group were compared using Fisher’s Exact Binomial Test with Bonferroni Correction Procedure. For the determination of the LOEL and NOEL the numbers of live young produced per adult over the duration of the test (excluding any parents which died accidentally or inadvertently) for the control and each test group were compared using Step-down Jonckheere-Terpstra Test incorporating Shapiro-Wilks test on normal distribution and Levene’s test on variance homogeneity. Results from the control and each test group’s Daphnia length data were compared using Williams Multiple Sequential t-test Procedure incorporating Shapiro-Wilks test on normal distribution and Levene’s test on variance homogeneity. All statistical analyses were performed using the ToxRatPro software package (TOXRAT).

Table 1 - Summary of results

Endpoint		Concentration (mg/L LR WAF)	95% Confidence Limits (mg/L LR WAF)
Immobilization	EL10	Not determined	Not applicable
	EL20	Not determined	Not applicable
	EL50	Not determined	Not applicable
	No Observed Effect Loading Rate	100	-
	Lowest Observed Effect Loading Rate	Not determined	-
Reproduction	EL10	Not determined	Not applicable
	EL20	Not determined	Not applicable
	EL50	Not determined	Not applicable
	No Observed Effect Loading Rate	100	-
	Lowest Observed Effect Loading Rate	Not determined	-
Body Length	EL10	>100	Not determined
	EL20	Not determined	Not applicable
	EL50	Not determined	Not applicable
	No Observed Effect Loading Rate	32	-
	Lowest Observed Effect Loading Rate	56	-

Table 2 - Range-finding Test Data

Nominal Loading Rate (mg/L)	Percentage Immobilization After 10 Days Exposure	Sub-lethal Effects	Total Number of Live Young in First Brood
Control	0	None	80
10	0	None	87
100	0	None	74

 

Table 3 - Summary of Findings Following the Exposure of Daphnia magna for 21 Days

Nominal Loading Rate (mg/L WAF)	Parental (P1) Generation Mortalities (Initial Population = 10)			Total Number of Live Young	Number of Live Young from Parents Surviving to the End of the Test		Number of Live Young at the End of the Test Excluding Parents that were Accidental or Inadvertent Mortalities
	Total	Accidental	Inadvertent		Total	Average per Parent	Total	Average per Parent
Control	0	0	0	1386	1386	139	1386	139
10	1	0	1	1319	1319	147	1319	147
18	0	0	0	1444	1444	144	1444	144
32	0	0	0	1569	1569	157	1569	157
56	0	0	0	1541	1541	154	1541	154
100	0	0	0	1598	1598	160	1598	160

 

Table 4 - Summary of Daily Observations

Day	Nominal Loading Rate (mg/L)
	Control		10		18		32		56		100
	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young
1	10	0	10	0	10	0	10	0	10	0	10	0
2	10	0	10	0	10	0	10	0	10	0	10	0
3	10	0	10	0	10	0	10	0	10	0	10	0
4	10	0	10	0	10	0	10	0	10	0	10	0
5	10	0	10	0	10	0	10	0	10	0	10	0
6	10	0	10	0	10	0	10	0	10	0	10	0
7	10	0	9	0	10	0	10	0	10	0	10	0
8	10	0	9	0	10	0	10	0	10	0	10	0
9	10	138	9	117	10	139	10	140	10	138	10	126
10	10	0	9	0	10	0	10	0	10	0	10	1
11	10	32	9	0	10	25	10	52	10	73	10	159
12	10	208	9	222	10	176	10	222	10	168	10	108
13	10	0	9	0	10	0	10	0	10	0	10	0
14	10	34	9	0	10	28	10	54	10	49	10	76
15	10	284	9	344	10	346	10	367	10	316	10	322
16	10	0	9	0	10	0	10	0	10	0	10	0
17	10	43	9	0	10	3	10	68	10	47	10	78
18	10	278	9	304	10	345	10	311	10	340	10	335
19	10	0	9	0	10	2	10	0	10	0	10	0
20	10	0	9	0	10	0	10	28	10	54	10	0
21	10	369	9	332	10	380	10	327	10	356	10	393
TOTALS	10	1386	9	1319	10	1444	10	1569	10	1541	10	1598

Table 5 - Body Length of Surviving Adults at Day 21

Nominal Loading Rate (mg/L)	IndividualDaphniaLengths (mm)
	1	2	3	4	5	6	7	8	9	10
Control	4.0	4.5	4.0	3.9	3.9	4.0	3.9	3.8	4.1	4.1
10	4.2	4.0	3.8	4.2	-	4.0	4.0	3.9	3.8	3.8
18	4.0	4.0	3.9	4.1	4.0	3.8	3.8	3.5	4.0	4.0
32	3.9	3.9	4.0	3.9	3.9	4.0	4.1	3.7	3.8	3.9
56	3.8	3.8	4.1	4.0	4.0	3.3	3.8	3.4	3.5	4.0
100	3.6	3.7	3.3	3.6	4.0	3.5	3.7	3.8	3.8	4.0

- No measurement, Daphnia died during the test

Table 6 - Validation Criteria

The following validation criteria were achieved during the test:

	Required	Actual
Control mortality	≤20%	0%
Mean number of live young per surviving adult (control group)	≥60 after 21 days	138.6
Coefficient of variation for control group*	≤25%	8.3%
No ephippia produced	0	0
Dissolved oxygen	>3 mg O2/L	≥7.8 mg O2/L
pH (control group)	6 to 9 variation ≤ 1.5	7.6 to 8.6 1.0

*Based on total number of living offspring per parent animal alive at the end of the test

Validity criteria fulfilled:

yes

Conclusions:

Table one summarises the results after exposure of Daphnia magna to the test item based on the nominal test concentrations.

Description of key information

A study was performed to assess the chronic toxicity of the registered substance to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, “Daphnia magna Reproduction Test” referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as Water Accommodated Fractions (WAFs).

Chemical analysis of the fresh test preparations on Days 0, 6, 13 and 20 showed measured test concentrations to range from less than the limit of quantification (LOQ), which was determined to be 0.00074 to 0.058 mg/L. Chemical analysis of the aged test preparations on Days 1, 7, 14 and 21 showed measured test concentrations to range from less than the LOQ to 0.054 mg/L. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Effects on Reproduction:
After 21 days there were no statistically significant differences between the control and all the test groups in terms of the number of live young produced per adult.

Lethal Effects on the Parental Generation (P1): No mortalities occurred at the control, 18, 32, 56 and 100 mg/L loading rate WAF groups throughout the test.

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L