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Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-02-06 to 2006-03-09
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study without restrictions. The study was conducted according to the OECD Guideline 202; Part 2, and in compliance with GLP principles.
Reason / purpose for cross-reference:
read-across: supporting information
according to guideline
other: OECD Guideline 202, Part 2.
GLP compliance:
Analytical monitoring:
Details on sampling:
On the basis of results of both TOC and GCMS analysis of dissolved CTO components, accomplished with CTO Sample 3 during acute toxicity testing with Alga (OECD 201, STZ No. 13-05-001), Daphnia (OECD 202, Part 1, STZ No. 13-05-002) and Fish (OECD 203, STZ No. 13-05-003), study sponsor Arizona Chemical, it was concluded that both TOC and GCMS analysis would not be applicable at a test loading rate of 1 mg/L, because the methods were insufficiently sensitive to quantify the concentration of dissolved test substance in the WAFs.
Details on test solutions:

- Method: The test substance was comprised of many chemicals, some of which have low water solubility. The exposures were therefore carried out using WAFs (Water Accommodated Fractions) obtained by stirring 1 mg/L loading rate with Daphnia medium for 24 h. The stock solution was prepared in Di-ethyl-ether with a corresponding loading rate in the WAF of 1 mg/L in 2 L glass beakers. Once the solvent was allowed to evaporate, the Daphnia medium was transferred into the beaker, stirred for 24 h and then allowed to stand for 1 h in order for phase separation to take place. To ensure that emulsification and dispersion of undissolved parts of the test materials did not occur, the surface vortex developed by stirring was set at ~ 10% of the total depth of the water. The beakers were equipped with a glass tube that allowed the separation of the water phase by siphoning. The duration of the stirring and phase separation phases of WAF preparation were investigated in a preliminary study In a preliminary with a test article of a comparably low water solubility (Sylvablend TM PF 40, STZ No. 01-05-001; study sponsor Arizona Chemical).

- Controls: culturing medium.

- Evidence of undissolved material: The test substance was not fully soluble; to avoid adverse physical effects of undissolved or emulsified test material on the animals, the test was carried out with aqueous extracts (WAFs).
Test organisms (species):
Daphnia magna
Details on test organisms:

- Common name: Daphnia

- Clone: 5

- Source: German Environmental Ministry, Berlin

- Age of parental stock:
- Feeding during test

- Food type: algae Desmodesmus subspicatus

- Amount: 0.1-0.2 mg C/L and Daphnia/day

- Frequency: daily


- Acclimation conditions: same as test - cultivated in a climate controlled room with illumination 14 h per day in culturing medium. Culturing medium is made up of stock solutions in deionised water:

CaCl2 x 2 H2O: 11.76 g/L
KCl: 0.23 g/L
MgSO4 x 7 H2O: 4.93 g/L
NaHCO3: 2.59 g/L

25 ml aliquots of each stock solution were mixed and then topped up to 1 L with deionised water. The water was then aerated until the concentration of oxygen complied with the saturation value of atmospheric oxygen.
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
Post exposure observation period:
231 ppm (as CaCO3)
Test temperature:
20 ± 2 °C
Dissolved oxygen:
7.8-8.3 mg/L
Nominal and measured concentrations:
Nominal: control and 1 mg/L.

Measured concentration: GCMS and TOC analytical methods were not sensitive enough to quantify the test substance in the WAFs at the loading rate of 1 mg/L.

The results of the test were reported and interpreted with reference to nominal loading rates.
Details on test conditions:

- Test vessel: beakers

- Material, size, fill volume: glass, 50 mL filled with 20 mL of test medium.

- Aeration: none

- Renewal rate of test solution: 48 h

- No. of organisms per vessel: 1

- No. of vessels per concentration: 10

- No. of vessels per control: 10

- A vehicle control was not required because the vehicle (di-ethyl-ether) was only used to facilitate adding the test substance to the WAF preparation vessel, after which it was evaporated off prior to adding the dilution water.


- Total organic carbon: 0.23 mg/L

- Alkalinity: 0.8 mmol/L

- Ca/Mg ratio: 4:1, total of 2.5 mmol/L

- Na/K ratio: 10:1

- Culture medium different from test medium: no

- Intervals of water quality measurement: every 48 h.


- Adjustment of pH: no

- Photoperiod: 16 h light, 8 h darkness

- Light intensity: Gro-Lux fluorescent tubes

EFFECT PARAMETERS MEASURED: Effects on development, mortality (immobilisation), growth and reproduction (total number of live offspring) of the Parental generation (P) are assessed. Additional test parameters are the date of first appearance of young Daphnia (F1 = Filial generation), appearance of aborted eggs, ephippia and dead Daphnia of the F1 generation and any other observed adverse effects of the test article on Daphnia.


Reference substance (positive control):
21 d
Dose descriptor:
Effect conc.:
>= 1 mg/L
Nominal / measured:
Conc. based on:
other: Water Accomodated Fractions (WAFs)
Basis for effect:
other: reproduction, growth and mortality
Details on results:
- Mortality of parent animals: 0% in control and 10% in at 1 mg/L during 21 d. 10% mortality is below the limit of 20% set in the guideline for determining a significant effect.

- No. of offspring produced per day per female: 100.9 ± 21.4 in control vessels, 101.7 ± 24.8 at loading rate 1 mg/L (mean ± SD, standard deviation); see table 3 for details.

- Body length and weight of parent animals: 4.67 ± 0.15 in control vessels, 4.74 ± 0.13 (mean ± SD, standard deviation); see table 4 for details.

- Number of males and females: no males were observed in the test.

- Time to first brood release or time to hatch: 10 days in control and at 1 mg/L loading rate.

- Other biological observations:
- day 19: one aborted egg in control vessel 8, at test loading rate 1 mg/L there were 38 aborted eggs in vessel 4, 2 aborted eggs in vessel 5, 39 aborted eggs in vessel 6.
- day 20: one day animal of the filial generation (F1) in control vessel; at test loading rate 1 mg/L one aborted egg and 15 dead animals of F1 in vessel 2, 9 dead animals from F1 were found in vessel 3, 2 aborted eggs were found in vessel 4, one dead animal from F1 was seen in vessel 7, 9 dead animals of F1 were found in vessel 8 where 20 animals from F1 showed slower swimming movements.

- Effect concentrations exceeding solubility of substance in test medium: it is possible that at the loading rate at which the water-accommodated fraction was prepared the solubility of some constituents of the test substance may have been exceeded.
Reported statistics and error estimates:
The numbers of live offspring of the Filial generation (F1) derived from the surviving animals of the Parental generation (P) were analysed statistically using Dunnett’s test and Bartlett’s test.

Table 3. Number of live offspring of the filial generation (F1) of the control and 1 mg/L test loading rate during the 21 d test; mean value, standard deviation (SD) and variation coefficient (Vc).

 Vessel  F1 (control)  F1 (1 mg/L)
 95  136
 2  109  88
 3  117  100
 4  119  80
 5  100  71
 6  70  78
 7  119  139
 8  88  113
 9  65  110
 10  127   - *
 Total  1009  915
 Mean  100.9  101.7
 SD  21.36  24.82
Vc  21.17  24.42
 Variance  456.3  616.3

* = total derived from 9 surviving F1 animals at 1 mg/L test loading rate.

The number of live offspring of the Filial generation (F1), derived from the surviving animals of the Parental generation (P), in the control and 1 mg/L test loading rate were not significantly different.

Table 4. Body length (mm) of living animals of the Parental generation (P) at the end of the 21 d test; mean-value, standard deviation (SD) and variation coefficient (Vc).

 Vessel  Control  1 mg/L
 1  4.70  4.90
 2  4.50  4.80
 3  4.65  4.85
 4  4.85  4.80
 5  4.80  4.60
 6  4.55  4.70
 7  4.75 4.55 
 8  4.90  4.60
 9  4.55  4.85
 10  4.45   -*
 Mean Value  4.67  4.74
 SD  0.15  0.13
 Vc  3.32  2.73

* = animal died on day 6.

Mean body lengths of surviving animals of the Parental generation (P) were comparable in the control and at 1 mg/L loading rate after 21 d.

Validity criteria fulfilled:
A 21 day NOELR of ≥1 mg/L has been determined for the effects of the test substance on reproduction, growth and mortality of Daphnia magna.

Description of key information

NOELR (21day): ≥1 mg/L, reproduction, growth and mortality Daphnia magna, reliability 1 (read-across from Crude Tall Oil).

DTO is a UVCB substance and compositional information has been used in conjunction with validated QSARs to derive long-term No Observed Effect Concentrations (NOECs) for its constituent blocks. Calculation of PNECs for the aquatic compartment will be based on data for the constituent blocks rather than on data for the whole substance. Further testing for long-term toxicity to invertebrates will therefore not contribute to the database that is required to complete the assessment of the substance.

Key value for chemical safety assessment

Additional information

There are no data for long-term toxicity of the substance to aquatic invertebrates. However reliable results are available for the closely-related substance, Crude Tall Oil (CAS No. 8002-26-4). A 21 day NOELR of ≥1 mg/L has been determined for the effects of the substance on reproduction, growth and mortality of Daphnia magna. The result was obtained in a test conducted on a sample of CTO that showed the highest toxicity in short-term tests with D. magna.

Read-across of long-term toxicity data for aquatic invertebrates from CTO to DTO is justified on the grounds that all the chemical constituents that are present in DTO are present in CTO i.e. fatty acids, resin acids, polymers and neutral compounds containing alcohols, hydrocarbons etc. The chemical constituent blocks of which they are comprised are compositionally very similar and are made up of constituents with common functional groups and properties. As a result the substances share very similar physicochemical properties both in terms of their bulk properties and the properties of the constituent blocks. CTO does contain some constituents that are not present in DTO and for this reason read-across from DTO to CTO is not considered appropriate. However for the purposes of read-across from CTO to DTO this is only likely to make the outcome conservative i.e. if anything the hazard of DTO is likely to be overestimated. A more detailed description of the technical justification for read-across is given in Section 1.4 of the CSR.