**5. Elimination of** *Ascaris* **ova in municipal sludge**

#### **5.1. Sampling: experimental procedure**

Compared to bacterial analyses of influent and effluent sewage sludge samples, *Ascaris* ova analyses required much larger volumes of biosolids (sewage sludge). Thus, several 1 l samples of sludge were collected in sterile glass bottles. Some samples were obtained as influent samples, while others were obtained as effluent samples, transferred on ice immediately after collection, and transported to the microbiology laboratory for prompt *Ascaris* ova counts.

#### **5.2. Sample analysis: experimental procedure**

of THB survived the treatment, only 0.85 ± 0.23% and 1.85 ± 0.65% of the initial populations of TC and FC survived, respectively. At doses of 13.2 kGy and above, neither TC bacteria nor FC were detected. Nevertheless, at a 13.2 kGy irradiation dose, 8.9 ± 1.3% of THB from the initial population survived the treatment. At a dose of 25.7 kGy and above, no significant THB from the initial population were left in treated sewage sludge samples [7]. **Table 3** summarizes bacterial counts per gram of sludge dry weight at different electron beam doses. From these results, D10-values were determined as 8.94, 3.16, and 3.17 kGy for THB, TC, and FC respec‐ tively. D10-values are defined as doses necessary to kill 90% of the bacterial populations in the sample for irradiation conditions applied, or the dose needed to reduce the bacterial popula‐ tion by a factor of 10. A close look at **Table 2** shows that dose 6.7 kGy reduces the FC counts to 180 colony forming unit (CFU) per gram of sludge dry weight, a count that is within the Environmental Protection Agency (EPA) norm to classify such treated municipal sewage sludge as class A sludge utilizable for land application in agriculture [9]. However, from the D10-value determined for FC, based on initial population of FC in influent samples, the dose required to convert this sludge to class A was estimated to be 4.5 kGy. Although no previous work similar to this one is known to perform a comparison with our estimated D10-value, nevertheless, water-based and surface membrane *Bacillus* spore killing D10-values were reported to be 1.3 and 1.53 kGy, respectively [10, 11]. These values are about twice lower than the 3.17 kGy determined in our case. This difference could be attributed to the presence of a large amount of organic and inorganic materials that make our sample relatively thick and

slightly viscous compared to water and a surface membrane.

**Table 3.** Bacterial counts in sludge samples at different irradiation doses [7].

**5. Elimination of** *Ascaris* **ova in municipal sludge**

**5.1. Sampling: experimental procedure**

**Total heterotrophic bacteria**

0.0 15.00% 1.4 × 106 1.7 × 105 2.0 × 104 2.7 25.75% 8.6 × 105 8.2 × 104 1.5 × 104 6.7 20.46% 3.2 × 105 9.3 × 102 1.8 × 102 13.2 12.29% 4.5 × 104 0.0 0.0 25.7 3.67% 1.3 × 104 0.0 0.0 30.7 3.25% 6.1 × 102 0.0 0.0

Compared to bacterial analyses of influent and effluent sewage sludge samples, *Ascaris* ova analyses required much larger volumes of biosolids (sewage sludge). Thus, several 1 l samples

**Total coliforms**

Counts (CFU) per gram of dry weight

**Fecal coliforms**

**weight (gram) percent**

**Dose (kGy) Sluge dry**

240 Radiation Effects in Materials

From each 1 l sample, 500 ml of well-mixed sludge was transferred in a blender, then 200 ml of sterile water was added, and the mixture was blended for 1 min at high speed. The blended mixture was transferred to a 1-l tall beaker to which 1% 7× detergent was added in order to reach 900 ml final volume. The same procedure was repeated for the second half the sludge sample, and the homogenized mixtures were combined and allowed to settle overnight in a cold (4°C) room or in a refrigerator. At this stage, some floating materials may be observed; therefore, stirring occasionally the mixture with a wooden applicator has shown to help settle the material. The supernatant was discarded by vacuum aspirating it to right above the layer of biosolids. The settled sediments were then transferred into a blender to which 500 ml of sterile water, blended again for 1 min at high speed, and transferred to a beaker. The blender was rinsed, and 1% 7× detergent was added to reach 900 ml final volume. Samples were allowed to settle for 2 h at 4°C after which the supernatant was discarded by vacuum aspirating it to right above the layer of biosolids. The biosolids were resuspended into 300 ml of 1% 7× detergent and stirred for 5 min using a magnetic stirrer. Homogenized sample was then strained through a 50 mesh (300 μm) sieve placed in a funnel over a beaker. Samples were washed through the sieve with a spray of 1% 7× detergent from a spray bottle. The sample volume in the beaker was adjusted to 900 ml by adding the necessary amount of 1% 7× detergent and allowed to settle for 2 h at 4°C. The supernatant was discarded using a vacuum, while the sediments were mixed and equally distributed in 50-ml centrifuge sterile tubes. In each tube, the sample volumes were adjusted to 50 ml with sterile water and centrifuged for 10 min at 1000×g. The supernatant was then discarded, and the pellet (biosolids) that should not exceed 5 ml was resuspend in 10 ml of MgSO4 (specific gravity 1.2). Each tube was vortexed for 2 min, and more MgSO4 was added to each tube to reach a volume of 50 ml. The tubes were then centrifuged for 10 min at 1000×g. The top 25–35 ml of supernatant of each tube was poured through a 400 mesh (38 μm) sieve supported in a funnel over a beaker. Biosolids retained on the sieve were washed, rinsed, and collected into a 100 ml beaker. The suspension of biosolids was then transferred into 15 ml centrifuge tubes. Tubes were centrifuged for 3 min at 800×g, and supernatants were discarded. If the previous step generated more than one tube for one initial sample, the sediments should be transferred into one single 15 ml tube and the centri‐ fugation step repeated. Finally, after discarding the supernatant, the biosolids were resus‐ pended in 4 ml 0.1 N H2SO4. The vials were incubated at 26°C for 3 weeks. After 24 days of incubation, when the majority of the controls were fully embryonated, samples were ready to be examined microscopically (10×) using a Sedgwick Rafter cell to enumerate the detected ova. Ova were classified as either nonviable (unembryonated) or viable (embryonated to the first, second, or third larval stage, those with the potential to become adult *Ascaris*). The percent moisture of the sample was determined by analyzing a separate portion of the sample, so that the final calculation of ova per gram dry weight could be determined. This was done by measuring the weight of the sludge samples before and after incubating at 45°C for 4 days, until dry by observation. Categories of ova per 4 grams per weight were calculated in the following manner:

Ova/g dry wt = (NO) × (CV) × (FV) / (SP) × (TS)

where NO = no ova, CV = chamber volume (=1 ml), FV = final volume in ml, SP = sample processed in ml or g, TS = % total solids.

#### **5.3. Results and discussion**

In order to determine *Ascaris* ova viability per four grams of sludge dry weight, the average dry weight of untreated sewage sludge samples was first determined. Hence, the dry weight of untreated sewage sludge samples was 15 ± 3% total solids. On average, untreated sewage sludge contained 312 ± 24 *Ascaris* ova per four grams of dry weight. **Figure 6** shows percentages of *Ascaris* ova viability after treatment of sewage sludge samples with electron-beam doses of 2.7, 6.7, 13.2, and 25.7 kGy.

**Figure 6.** Effect of electron beam irradiation on *Ascaris* ova survival in municipal sewage sludge. Percentages of *Ascaris* ova survival after irradiation of samples [7].

Similar to bacterial counts, the results indicate that the viability of *Ascaris* ova decreases in a dose-dependent manner [7] with a D10-value of 7.93 kGy. At dose 2.7 kGy, 23 ± 8% (72 ± 24 *Ascaris* ova per four grams of dry weight) of viable ova survived the treatment, while only 11 ± 1.6% (34 ± 4 *Ascaris* ova per four grams of dry weight) ova survived at dose 6.7 kGy. At dose 13.2 kGy, the survival rate dropped to 2 ± 0.03% (6 ± 1 *Ascaris* ova per four grams of dry weight). No *Ascaris* ova were detected in sewage sludge samples irradiated at 25.7 kGy. However from our counts, we estimated the electron-beam dose 14.5 kGy to be necessary to obtain a sewage sludge containing less than one *Ascaris* ova per four grams of sludge dry weight, meaning that the dose of 25.7 kGy applied during our experiments was high enough to achieve a class A sludge. Indeed, according to EPA standards, to be considered class A, sewage sludge must contain less than one *Ascaris* ovum per four grams of sludge dry weight [9].

until dry by observation. Categories of ova per 4 grams per weight were calculated in the

where NO = no ova, CV = chamber volume (=1 ml), FV = final volume in ml, SP = sample

In order to determine *Ascaris* ova viability per four grams of sludge dry weight, the average dry weight of untreated sewage sludge samples was first determined. Hence, the dry weight of untreated sewage sludge samples was 15 ± 3% total solids. On average, untreated sewage sludge contained 312 ± 24 *Ascaris* ova per four grams of dry weight. **Figure 6** shows percentages of *Ascaris* ova viability after treatment of sewage sludge samples with electron-beam doses of

**Figure 6.** Effect of electron beam irradiation on *Ascaris* ova survival in municipal sewage sludge. Percentages of *Ascaris*

Similar to bacterial counts, the results indicate that the viability of *Ascaris* ova decreases in a dose-dependent manner [7] with a D10-value of 7.93 kGy. At dose 2.7 kGy, 23 ± 8% (72 ± 24 *Ascaris* ova per four grams of dry weight) of viable ova survived the treatment, while only 11 ± 1.6% (34 ± 4 *Ascaris* ova per four grams of dry weight) ova survived at dose 6.7 kGy. At dose 13.2 kGy, the survival rate dropped to 2 ± 0.03% (6 ± 1 *Ascaris* ova per four grams of dry weight). No *Ascaris* ova were detected in sewage sludge samples irradiated at 25.7 kGy. However from

following manner:

242 Radiation Effects in Materials

Ova/g dry wt = (NO) × (CV) × (FV) / (SP) × (TS)

processed in ml or g, TS = % total solids.

**5.3. Results and discussion**

2.7, 6.7, 13.2, and 25.7 kGy.

ova survival after irradiation of samples [7].
