Naphthenic acids

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:

2017-10-29 to 2017-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Appearance: yellow/amber viscous liquidSource and lot/batch No.of test material: 2017787NA12
- Expiration date of the lot/batch: 2019-07-31 (expiry date)
- Purity test date: 2017-07-27 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle:
* water: 88.1 mg/L at 20°C
* hydrocarbon solvents: soluble

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples for analysis were taken from all test concentrations and the control. Samples were taken at the beginning and at the end of two intervals of 48 hours (nominal days 8 and 10, 15 and 17) and one interval of 72 hours (nominal days 0 and 3). At the end of the refreshment period, the replicates were pooled at each concentration before sampling.volumes of 2 mL were taken.
- Sample storage conditions before analysis: the samples were stored in a freezer (=<- 15°C) until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with loading rates individually prepared at loading rates ranging from 0.22 to 100 mg/L. A two-day period of magnetic stirring was applied to accelerate dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of approximately one hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning1 and used as test concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All test solutions were clear and colorless at the end of the preparation procedure.
Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water-flea
- Source: in-house laboratory culture with a known history
- Age at study initiation (mean and range, SD): <24h (from parental daphnids of more than 2 weeks old)
- Method of breeding: 250 newborn daphnids (i.e. less than 3 days old) are placed into 5 litres of medium in an all-glass culture vessel. Medium is M7. After 7 days of cultivation half of the medium is renewed twice a week.
- Feeding during test: Twice daily an amount of 0.25 ml of a Chlorella sp. suspension. On weekend days an amount of 0.50 ml was added in one single feed. This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211.

ACCLIMATION
- Acclimation period: max. 4 weeks
- Acclimation conditions (same as test or not): yes
- Type and amount of food: suspension of fresh water algae.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

161-179 mg/L as CaCO3

Test temperature:

19-21°C

pH:

7.7-8.3

Dissolved oxygen:

7.4-9.9 mg O2/L

Nominal and measured concentrations:

Range finder:
Nominal concentrations (mg/L): Control, 1.0, 10, 100
Measured concentrations (mg/L) Control, 1.10, 11.7, 107

Final test:
Nominal concentrations (mg/L): Control, 0.22, 0.46, 1.0, 2.2, 4.6
Measured concentrations (mg/L) Control, 0.15, 0.36, 0.84, 1.9, 4.1

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass covered with a Perspex plate
- Material, size, headspace, fill volume: 60 mL, glass beaker
- Aeration: not during the test
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus.Protoplasma 154, 25-33).

OTHER TEST CONDITIONS
- Photoperiod: 16h light daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Every workday the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed. Every workday the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.

TEST CONCENTRATIONS
- Final test
- 0, 0.22, 0.46, 1.0, 2.2, 4.6 mg/L
- Range finding study
- Test concentrations: 0, 0.1, 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 4.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 4.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

4.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of control: 10%
- Other adverse effects control: signifcant effect on length (4.9-6.4% reduction in length after 21 days observed in exposed test organisms)
- Abnormal responses: none

Results with reference substance (positive control):

- Results with reference substance valid? no reference substance
- Mortality: no
- Parameter Naphthenic acids Average exposure concentration (mg/L)
NOEC for reproduction 4.1
EC10 and EC50 for reproduction >4.1
NOEC for mortality 4.1
EC10 for growth >4.1

Reported statistics and error estimates:

- No statistical analysis was performed on the survival of introduced daphnids as the observed effects did not exceeded 10%. This is below the mortality allowed by the guideline for the control treatment (i.e. 20%) and therefore, considered biologically not relevant.
The following statistical procedures were used to determine the NOEC for reproduction and growth:
* Data distribution: Shapiro-Wilk´s Test
* Homogeneity of variance: Levene´s Test (with Residuals)
* Differences between treatments and the control:
* Reproduction: Multiple Sequentially-rejective U-test After Bonferroni-Holm (Alpha is 0.050; one-sided smaller).
* Length: Williams Multiple Sequential t-test Procedure (Alpha is 0.050; one-sided smaller).
- EC-values for reproduction: No EC10 and EC50 values could be calculated as the observed effects were lower than 10%. All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

RANGE-FINDING TEST

 

Cumulative mortality and reproductive potency during the range-finding test:

 

	Naphthenics acids, WAF prepared at loading rate (mg/L)
Nominal	Control		1.0		10		100
day	A	B	A	B	A	B	A	B
0	0	0	0	0	0	0	0	0
1	0	0	0	0	0	0	4	4
2	0	0	0	0	0	0	5	5
3	0	0	0	0	0	0	5	5
4	0	0	0	0	0	0	5	5
5	0	0	0(2)	0(2)	0	1	5	5
6	0(3)	0(2)	0(4)	0(4)	1	4	5	5
7	0(5)	0(5)	0(5)	0(5)	1	4	5	5
8	0(5)	0(5)	0[19]	0[18]	1	4	5	5
9	0[33]	0[32]	0[4]	0[14]	1	4	5	5
10	0[17]	0[28], 1E	0[16]	0[11]	1(1)	4(1)	5	5

() number of parents with eggs in their brood pouch

[] number of offspring

E number of aborted eggs

 

No mortality was observed in the control and the WAF prepared at a loading rate of 1.0 mg/L. In the WAF prepared at 10 mg/L 50% of exposed daphnids did not survive the 10 day of exposure, while in the highest groups no survivors were present at day 2 of the exposure. First offspring were observed in the control treatment at day 9 of exposure, while in the lowest WAF on day 8.

In the WAF prepared at 10 mg/L no reproduction was observed. Only two of the 5 surviving daphnids were observed to have eggs in the brood pouch at the last day of the exposure. All surviving daphnids in this group were observed to be smaller than the control animals.

Despite the fact, that the onset of reproduction in the WAF of 1.0 mg/L was one day earlier than in the control treatment, the total number of offspring was less than half of what was observed in the control.

Based on these results, it was decided not to include the WAF of 10 mg/L in the final test and to set the range between 0.22 and 4.6 mg/L.

 

REPRODUCTION TEST

 

Nominal and mean measured exposure concentrations: 

Naphthenic acids loading rate (mg/L)	Mean exposure concentration (mg/L)			Average exposure concentration (mg/L)*
	Day 0-3	Day 8-10	Day 15-17
0.22	0.13	0.19	0.15	0.15(70)
0.46	0.33	0.42	0.35	0.36(70)
1.0	0.74	0.95	0.89	0.84(84)
2.2	1.8	2.0	1.9	1.9(87)
4.6	3.9	4.4	4.2	4.1(90)

* Between brackets : percentage recovery relative to nominal loading rate

 

Samples taken from all groups were analysed. The actual concentrations measured in the freshly prepared solutions were at the level of 78-110% of the nominal loading rates. Measured concentrations decreased to 60-77% of initially measured in the old solutions.

Mortality (immobility) of parental daphnids at the end of the test:

 

Naphthenic acids Average exposure concentration (mg/L)	Introduced	Mobile	Immobile	% Immobility
Control	20	18	2	10
0.15	10	9	1	10
0.36	10	9	1	10
0.84	10	10	0	0.0
1.9	10	10	0	0.0
4.1	10	10	0	0.0

 

 Two out of the twenty parental daphnids died during the test period in the control treatment. Hence, parental mortality did not exceed 20% in the controls. In the two lowest groups mortality of parental daphnids was 10% at the end of the test, while in the three highest groups no mortality was observed. As no dose-related response was observed, the reproduction was calculated per surviving parent.

Group mean cumulative number of juveniles per surviving parent and reduction of reproduction at the end of the test:

 

Naphthenic acids Average exposure concentration (mg/L)	Mean	Std. Dev.	N	%Reduction*
Control	181.1	22.17	18
0.15	179.2	24.77	9	1.0
0.36	181.4	22.67	9	-0.2
0.84	186.9	21.56	10	-3.2
1.9	179.4	25.59	10	0.9
4.1	191.8	25.00	10	-5.9

* Negative numbers indicate stimulation rather than reduction of reproduction

 

No delay in the reproduction was observed at any of the groups tested when compared with the untreated control.

On average, 181 offspring were produced per surviving daphnid in the control treatment. Responses recorded in test item treated groups were not related to the applied dose and ranged from 1.0% reduction to 5.9% stimulation

 

Incidence of immobilized offspring and aborted eggs:

 

Naphthenic acids Average exposure concentration (mg/L)	Immobile offspring	Aborted eggs
Control	3	3
0.15	1	13
0.36	5	0
0.84	5	1
1.9	4	0
4.1	0	4

A number of immobile offspring and aborted eggs were observed in this test. The appearance of the aborted eggs and immobile offspring was not dose-related.

Group mean body lengths (mm) and reduction of growth of parental daphnids at the end of the test:

 

Naphthenic acids Average exposure concentration (mg/L)	Mean (mm)	Std. Dev.	N	% Reduction
Control	4.60	0.338	18
0.15	4.32*	0.171	9	6.0
0.36	4.31*	0.115	9	6.4
0.84	4.38*	0.270	10	4.9
1.9	4.31*	0.249	10	6.4
4.1	4.35*	0.224	10	5.4

  * Statistically significant (p<0.05)

 

The reduction of body length ranged from 4.9 to 6.4% but was not dose-related. The observed reduction was found statistically different from the control treatment at all concentrations. Nevertheless, as no dose-related, it was considered irrelevant. Therefore, no NOEC was set for this end point, instead an EC10 value was given.

Exposure parameters :

 

Condition		Range
pH		7.7 – 8.3
Temperature measured in test vessels (°C)		19 – 21
Temperature measured continuously in control vessel (°C)		19 – 20
Dissolved oxygen concentration (mg O2/L)		7.4 – 9.9
Hardness (mg CaCO3)		161 – 179
Total dissolved organic carbon content (mg/L)		12
Light intensity (Lux)	Start test	819 – 892
	End test	795 - 866

 

The pH was maintained within the limits prescribed by the study plan (6.0-9.0, constant within 1.5 units).

The oxygen concentration complied with the requirements as laid down in the study plan (> 3 mg/L).

The temperatures complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Total hardness complied with the requirements as laid down in the study plan (>140 mg CaCO3 per liter).

The total dissolved organic carbon content of the M7 medium exceeded the value recommended by the guidelines (i.e. was > 2 mg/L). However, this was assumed not to influence the results of the study.

Discussion

The range-finding test showed that exposure to the WAF prepared at a loading rate of 10 mg/L causes 50% mortality of introduced daphnids after 10 days of exposure. Moreover, growth and reproductive potential of daphnids exposed to this treatment was significantly affected after such a short exposure period.

No effects on reproduction and survival of introduced daphnids was observed at the highest concentration tested in the reproduction test, which was a factor of 2.2 lower than the WAF of 10 mg/L. Therefore, the NOEC on survival and reproduction was 4.1 mg/L (WAF of 4.6 mg/L).

Although the growth was significantly affected at all concentrations tested, the observed effects were considered not relevant as being not dose-related. Moreover, the main endpoint of this study was reproduction and testing concentration range below the WAF of 2.2 mg/L would not yield any refinement of this endpoint. In addition it would be technically not feasible.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of naphthenic acids to Daphnia magna was determined in a 21 days semi-static test according to the OECD guideline 211.
Based on average measured exposure concentrations, the EC10 and EC50 for reproduction were determined to be >4.1 mg. The results of the test can be considered reliable without restriction.

Description of key information

In a long-term toxicity study to Daphnia magna according to OECD guideline 211 no effects were seen up to a concentration of 4.1 mg/L (unbounded NOEC and EC10).

In a study of Kinley et al (2016), Ceriodaphnia dubia, Hyallela azteca and Ceriodaphnia dilutus were exposed for 7 days to NA. The LC 50 were in the range of 2.8 -6.5 mg/L). As only survival was followed in this study, this information is only used as supporting information.